Kdm4 inhibitors

ABSTRACT

The present disclosure relates generally to compounds and methods for inhibiting the enzymatic activity of lysine demethylase 4 (KDM4) and treating cancer. Provided herein are substituted pyridine derivative compounds and pharmaceutical compositions comprising said compounds. The subject compounds and compositions are useful for inhibiting lysine demethylase 4. Furthermore, the subject compounds and compositions are useful for the treatment of breast cancer and the like.

RELATED APPLICATION

This application is a § 371 National Stage Application ofPCT/US2018/024624, filed Mar. 27, 2018, which claims priority benefit ofU.S. Provisional application No. 62/478,785, filed Mar. 30, 2017, andU.S. Provisional application No. 62/513,875, filed Jun. 1, 2017, both ofwhich are incorporated entirely herein for all purpose.

FIELD

The embodiments described herein relate to compounds and methods forinhibiting the activity of the enzyme lysine demethylase 4 (KDM4).

BACKGROUND

There remains a need for compounds and methods for treating cancers.

SUMMARY

The present embodiments provide compounds and methods for inhibiting theenzymatic activity of KDM4 and treating cancers. More specifically, anaspect of the present embodiments provides substituted pyridinederivatives that inhibit KDM4 (“KDM4(i)”) and exhibit unique preclinicalcharacteristics. At least one embodiment provides a potent pan-KDM4(i),Compound I, that specifically blocks the demethylase activity of KDM4A,4B, 4C, and 4D, but not that of other KDM family members. KDM4(i)anti-tumor properties were validated under conditions recapitulatingpatient tumors.

Another aspect of the present embodiments provides a method to screenKDM4(i) in triple-negative breast cancer stem-cells (BCSCs) preparedfrom individual patient tumors after neoadjuvant chemotherapy andpropagated in vitro. Limiting dilution orthotopic xenografts of theseBCSCs faithfully regenerated original patient tumor histology and geneexpression. In at least one embodiment, KDM4(i) as described hereinblocks proliferation, sphere formation, and xenograft tumor growth ofBCSCs prepared according to the method.

In another embodiment, KDM4(i) compounds abrogate expression of EGFR, adriver of therapy-resistant, triple-negative breast tumor cells viainhibition of the KDM4A demethylase activity. This activity isparticularly relevant in the context of BCSC from triple-negativetumors.

The present embodiments provide a unique BCSC culture system as a basisfor therapeutic compound identification, and demonstrate that KDM4inhibition is a therapeutic strategy for the treatment of cancersincluding triple-negative breast cancer.

DESCRIPTION OF THE DRAWINGS

FIG. 1A to FIG. 1H present data related to characterization of breastcancer stem cells (BCSC), and cell lines and xenografts derivedtherefrom. FIG. 1A shows representative growth data for alimiting-dilution assay of BCSC1 cell xenografts in immunocompromisedmice. FIG. 1B shows immunohistochemical analysis of the original BCSC1patient tumor and the BCSC1 xenograft tumor comprising hematoxylin andeosin (H&E), anti-CK8, anti-Ki67, anti-E-cadherin, and anti-vimentinstaining. Scale bar: 100 am. FIG. 1C shows anti-ER, anti-PR and HER2immunohistochemical staining of the original BCSC1 patient tumor, theBCSC1 xenograft tumor, and positive control sample. ER: estrogenreceptor; PR: progesterone receptor; HER2: human epidermal growth factor2; scale bar: 100 am. FIG. 1D provides an unsupervised hierarchicalcluster analysis of RNA microarray data presented as a clusterdendrogram. Samples are original patient tumors BCSC1, BCSC2, BCSC3, andBCSC4; derived BCSC1-4 lines; and BCSC1-4 xenograft tumors derived fromthe BCSC lines 1-4. FIG. 1E is representative pictures showing thecellular phenotype of BCSC1 cells cultured in 3D (top panel) and 2D(bottom panel) conditions. Scale bar, 100 am. FIG. 1F demonstrates thesphere-forming capacity of BCSC1 cells in a methylcellulose assay (n=3).Comparisons were made using a one-way ANOVA. Data represent means±s.e.m.*P<0.05, **P<0.01, ***P<0.001. FIG. 1G and FIG. 1H show representativeexpression patterns of cancer stem cell markers in BCSC1 cells, asanalyzed by FACS (n=3): FIG. 1G shows expression of CD24 and CD44markers; FIG. 1H shows expression of CD49f and EpCAM markers.

FIG. 2A to FIG. 2F demonstrate that KDM4 inhibitors (“KDM4(i)”) arepotent inhibitors of BCSC1 cells. FIG. 2A shows anti-KDM4A, antiKDM4B,anti-KDM4C, anti-KDM4D, and anti-tubulin western blots. Samples arelysates from HEK293T; HEK293T transfected with expression plasmidsexpressing KDM4A, KDM4B, KDM4C, or KDM4D; BCSC1; and BCSC2 cells. FIG.2B illustrates the formula/structure of a particular embodiment ofKDM4(i), Compound I. FIG. 2C shows a representative cell proliferationassay of BCSC1 cells cultured in absence and presence of Compound I(n=3). ●: vehicle; ▪: 10 nM Compound I; ▴: 50 nM Compound I; datarepresent means±s.d. FIG. 2D is a representative dose-response curve forKDM4(i) on BCSC1 cells (n=3). Data represent means±s.d. FIG. 2E showsthe 3D sphere-forming capacity of BCSC1 in an anchorage-independentgrowth assay in absence and presence of the indicated concentrations ofa KDM4(i) (n=3); 0: vehicle; 10: 10 nM Compound I; 50: 50 nM Compound I.FIG. 2F shows primary (1° spheres) and secondary (2° spheres)sphere-forming capacity of BCSC1 cells in Matrigel (n=3); 10: 10 nMCompound I; 50: 50 nM Compound I; comparisons made using one-way ANOVA;data represent means±s.e.m.*P<0.05, **P<0.01, ***P<0.001.

FIG. 3A to FIG. 3J shows that a KDM4(i) can target BCSC through EGFRregulation. FIG. 3A is a pie chart displaying the number of genes thatare differentially regulated in BCSC1 cells upon treatment with aKDM4(i): the transcriptome of BCSC1 cells with or without KDM(i)exposure (580 genes, p<1e-5): 254 upregulated genes and 326downregulated genes. FIG. 3B is a pie chart showing genomic distributionof KDM4A in BCSC1 cells as determined by ChIP-seq analysis: KDM4A peaks(172,692 peaks): 12.5% promoter; 40.5% intergenic; 41.0% intron; 3.3%exon; and 2.7% 3′UTR. FIG. 3C is a Venn diagram showing the intersectionand number of genes where KDM4A is present on the promoter region withgenes that are differentially regulated in BCSC1 cells upon treatmentwith a KDM4(i). A hypergeometric test calculated significance ofoverlaps (c; p<10-50). FIG. 3D illustrates KEGG pathways analyses(pathway enrichment analysis/common pathways) (pathways enriched for theset of 419 genes depicted in FIG. 3C). FIG. 3E reflects mRNA levelanalysis, more specifically a heat-map representing the mRNA levelsfound in BCSC1 cells cultured in absence (−) and presence (+) of aKDM4(i). The 37 direct target genes of KDM4A represent a gene signaturecommon for all the pathways represented in FIG. 3D. FIG. 3F is a bargraph of expression analyses in samples obtained from BCSC1 cellscultured in the absence (black bars) and presence (gray bars) of KDM4(i)(n=3). Data represent means±s.d. ***p<0.0001; **p<0.001 by two-tailedStudent's test. FIG. 3G and FIG. 3H are photos of anti-EGFR, anti-KDM4A,and anti-tubulin western blots generated from lysates of BCSC1 cells inthe absence (−) and presence (+) of a KDM4(i) (FIG. 3G); or BCSC1 cellstreated with shRNA control (Ctrl) or anti-KDM4A shRNA (FIG. 3H). FIG. 3Iis a graph showing meta-analysis of sequencing read density based onH3K9me3 ChIPseqs around KDM4A peaks in BCSC1 cells cultured in thepresence (gray) or absence (black) of a KDM4(i). A hypergeometric testwas done to calculate the significance of the overlaps; p<10-50). FIG.3J shows BCSC1 cells ChIP-Seq tracks analysis in the absence (dark) orpresence (light) of a KDM4(i). Normalized levels of H3K9me3 tracks atthe EGFR promoter.

FIG. 4A to FIG. 4F show that KDM4(i) inhibits xenograft tumor growthfrom BCSC1 cells. Mice bearing BCSC1 xenograft tumors were treated for21 consecutive days with either vehicle or a KDM4(i). FIG. 4A is a photoof representative xenograft BCSC1 tumors isolated from individualanimals after 21 day of treatment. FIG. 4B is a graph depictingdevelopment of tumors, measured in mm³, over time (n=11) (vehicle);(n=12) (a KDM4(i) treatment)). Data represent means±s.e.m. FIG. 4C showstumor weight at the end of experiment (n=1 (vehicle); n=12(KDM4(i)-treated)). Comparisons via one-way ANOVA.Data=means±s.e.m.*P<0.05, **P<0.01, ***P<0.001. FIG. 4D showsrepresentative images of tumors; and FIG. 4E is a bar graph of volumesize of tumor, in which data was obtained by ultrasound imagery at thestart of treatment (Day 0) and after 21 days of treatment (Day 21) (n=1(vehicle), n=12 (KDM4(i) treatment)). Comparisons were made using aone-way ANOVA. Data represent means±s.e.m.*P<0.05, **P<0.01, ***P<0.001.FIG. 4F is a bar graph showing expression analyses of BCSC1 xenografts.Samples were obtained from BCSC1 xenograft tumors of mice treated withvehicle (−) or a KDM4i (+KDM4(i)). Error bars, s.d.; biologicalreplicates (n=3). ***p<0.0001; **p<0.001 by two-tailed Student's test.

FIG. 5A to FIG. 5G demonstrates that BCSC2 cells and xenograftrecapitulate the original tumor patient. FIG. 5A graphs representativegrowth curves for a limiting-dilution/BCSC2 xenograft formation assay ofBCSC2 cells using BCSC2 xenografts in an immunocompromised mouse model.FIG. 5B shows photos of hematoxylin and eosin (H&E), anti-CK8,anti-Ki67, anti-E-cadherin, and anti-vimentin immunohistochemicalstaining. Samples are the original BCSC2 patient tumor and the BCSC2xenograft tumor. FIG. 5C shows photos of anti-ER, anti-PR and HER2immunohistochemical staining of the original BCSC2 patient tumor and theBCSC2 xenograft tumor. ER: estrogen receptor; PR: progesterone receptor;HER2: human epidermal growth factor 2; scale bar, 100 am. FIG. 5D showsrepresentative photos of BCSC2 cells cultured in 3D and 2D conditions.FIG. 5E is a bar graph showing sphere-forming capacity of BCSC2 cells inmethylcellulose assay (n=3). Comparisons via one-way ANOVA; datarepresent means±s.e.m.; *P<0.05, **P<0.01, ***P<0.001. FIG. 5F and FIG.5G are representative expression patterns of CSC markers CD24 and CD44(FIG. 5F); and CD49f and EpCAM (FIG. 5G) in BCSC2 cells analyzed by FACS(n=3).

FIG. 6A to FIG. 6F show that a KDM4(i) is a potent inhibitor of BCSC2cells. FIG. 6A is a graph reflecting a cell proliferation assay in whichBCSC2 cells were culture in the absence (●) or presence of 10 nm (▪) or50 nm (▴) KDM4(i). FIG. 6B is a graph depicting a representativedose-response curve for a KDM4(i) on BCSC2 cells (n=3). Data representmeans±s.e.m. FIG. 6C is a bar graph of BCSC2 sphere-formation inanchorage-independent growth assay, in absence (left bar) or presence of10 nM (middle bar) or 50 nM (right bar) of a KDM4(i) (n=3). FIG. 6D is abar graph of primary and secondary sphere-forming capacity of BCSC2cells in Matrigel. For the primary week, cells were culture in absence(−) and presence (50 nM) of a KDM4(i). Comparisons via one-way ANOVA.Data represent means±s.e.m.*P<0.05, **P<0.01, ***P<0.001. FIG. 6E andFIG. 6F are graphs representative of apoptosis assays of BCSC1 cells(FIG. 6E) and BCSC2 cells (FIG. 6F) in absence (Vehicle) or presence ofa KDM4(i), as analyzed by FACS (n=3). Data represent means±s.e.m.

FIG. 7A to FIG. 7K demonstrate that an embodiment of a KDM4(i) targetsBCSC2 through EGFR regulation. FIG. 7A is a Venn diagram of BCSC1 cellsand KDM4A locations, displaying the number of locations in control BCSC1cells (Ctrl (172,639)) and BCSC1 cells infected with an adenovirusexpressing an shRNA against KDM4A (KDM4A KD (3,215)) showing an overlapof 1110 locations. FIG. 7B and FIG. 7C show data of proliferation assaysof BCSC cells, more specifically representative proliferation of BCSC1(FIG. 7B) and BCSC2 (FIG. 7C) cells in absence (Vehicle) and presence of10 μM Erlotinib (n=3). Data represent means±s.e.m. FIG. 7D and FIG. 7Eare representative dose-response graphs of erlotinib exposure on BCSC1cells (FIG. 7D) and BCSC2 cells (FIG. 7E) (n=3). Data representmeans±s.e.m. FIG. 7F and FIG. 7G show BCSC1 (FIG. 7F) and BCSC2 (FIG.7G) 3D sphere-forming capacity in anchorage-independent growth assay inthe absence (left bar) and the presence of 1 μM (middle bar) or 10 μM(right bar) Erlotinib. Comparisons via one-way ANOVA;means±s.e.m.*P<0.05, **P<0.01, ***P<0.001. FIG. 7H and FIG. 7I showanti-EGFR, anti KDM4A, and anti-tubulin western blots. Samples arelysates from BCSC2 cells cultured in the absence and presence of aKDM4(i) (FIG. 7H); or treated with an shRNA control (Ctrl) or shRNAagainst KDM4A (FIG. 7I). FIG. 7J are two pie charts displaying genomicdistribution of H3K9me3 in BCSC1 cells in the absence (H3K9me3 peaks:141,722 peaks) or presence of KDM4(i) (H3K9me3 peaks: 144,266 peaks) asdetermined by ChIP-seq analysis: Vehicle: 6.2% promoter; 47.3%intergenic; 41.2% intron; 2.8% exon; 2.4% 3′UTR; +KDM4(i): 6.2%promoter; 47.7% intergenic; 40.7% intron; 2.9% exon; 2.4% 3′UTR. FIG. 7Kis a Venn diagram showing number and intersection of KDM4A and H3K9me3locations in BCSC1 cells cultured in the absence or presence of aKDM4(i). A hypergeometric test was done to calculate the significance ofthe overlaps (i; p<10⁻⁵⁰).

FIG. 8A to FIG. 8G demonstrate that a KDM4(i) inhibits xenograft tumorgrowth in mice bearing BCSC2 xenograft tumors treated for 21 consecutivedays with either vehicle or a KDM4(i). FIG. 8A is photos ofrepresentative xenograft BCSC2 tumors isolated from individual animalsafter 21 day of treatment. FIG. 8B is a graph showing development oftumors (measured in mm³) (n=6). Data represent means±s.e.m. FIG. 8C is abar graph of tumor weights at the end of the 21-day experiment (n=6).Comparisons via one-way ANOVA; data represent means±s.e.m. *P<0.05,**P<0.01, ***P<0.001. FIG. 8D shows representative images of tumors; andFIG. 8E is a bar graph of volume quantification of all tumors obtainedby ultrasound imagery at the start of treatment (Day 0) and after 21days of treatment (Day 21) (n=6). Comparisons via one-way ANOVA; datarepresent means±s.e.m. *P<0.05, **P<0.01, ***P<0.001. FIG. 8F and FIG.8G show body weight of mice bearing BCSC1 xenograft tumors (FIG. 8F) orBCSC2 xenograft tumors (FIG. 8G) over the treatment time-span of 21consecutive days with either vehicle or a KDM4(i). Data representmeans±s.e.m.

FIG. 9 presents a series of illustrations related to KGM4 structure andfunction. (A) is a schematic structure of four KDM4 proteins. The JmjNdomain is required for the activity of the JmjC catalytic center. (B)shows modes of KDM4 function as demethylases or independent of enzymaticactivity. (C) shows SDH, FH and IDH in the Krebs cycle. Succinateaccumulates upon SDH or FH mutation, while neomorphic IDH mutations leadto 2-hydroxyglutarate production. This figure is reproduced from Berry &Janknecht, KDM4/JMJD2 Histone Demethylases: Epigenetic Regulators inCancer Cells, 73(10) Cancer Res. 2936 (2013).

DETAILED DESCRIPTION

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Throughout this specification, unless otherwise indicated, “comprise,”“comprises” and “comprising” are used inclusively rather thanexclusively, so that a stated integer or group of integers may includeone or more other non-stated integers or groups of integers. The term“or” is inclusive unless modified, for example, by “either.” When rangesare used herein for physical properties, such as molecular weight, orchemical properties, such as chemical formulae, all combinations andsub-combinations of ranges and specific embodiments therein are intendedto be included. Other than in the operating examples, or where otherwiseindicated, all numbers expressing quantities of ingredients or reactionconditions used herein should be understood as modified in all instancesby the term “about.” The term “about” when referring to a number or anumerical range means that the number or numerical range referred to isan approximation within experimental variability (or within statisticalexperimental error), and thus the number or numerical range may varybetween 1% and 15% of the stated number or numerical range, as will bereadily recognized by context.

Unless otherwise defined, scientific and technical terms used inconnection with the formulations described herein shall have themeanings that are commonly understood by those of ordinary skill in theart. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.

The embodiments described herein provide therapy particularly indicatedwhen the disease state of the subject (e.g., cancer or neoplasticdisease) is associated with epigenetics or the epigenetic state of thesubject.

By way of background, epigenetics is the study of heritable changes ingene expression caused by mechanisms other than the underlying DNAsequence. Molecular mechanisms that play a role in epigenetic regulationinclude DNA methylation and chromatin/histone modifications. The genomesof eukaryotic organisms are highly organized within the nucleus of thecell. Tremendous compaction is required to package the 3 billionnucleotides of the human genome into the nucleus of a cell. Chromatin isthe complex of DNA and protein that makes up chromosomes. Histones arethe major protein component of chromatin, acting as spools around whichDNA winds. There are six classes of histones (HI, H2A, H2B, H3, H4, andH5) organized into two groups: core histones (H2A, H2B, H3, and H4) andlinker histones (HI and H5). The basic unit of chromatin is thenucleosome, which consists of about 147 base pairs of DNA wrapped aroundthe core histone octamer, consisting of two copies each of the corehistones H2A, H2B, H3, and H4. Changes in chromatin structure areaffected by covalent modifications of histone proteins and bynon-histone binding proteins. For example, DNA methylation, acetylation,and other post-translational modifications of the nucleosome histoneproteins alter chromatin organization and gene expression withoutaltering the underlying DNA sequence. Thus, alterations in the cellularepigenetic environment, and not only primary genetic mutations, play animportant role in tumor formation, progression and resistance totreatment, because epigenetic modification may influence if, when, orwhere specific genes are expressed. Chaidos et al., 6 Ther. Adv.Hematol. 128 (2015). Epigenetic modification is a dynamic and reversibleprocess that is written, erased, and read by various enzyme families.Several classes of enzymes are known which modify histones at varioussites. Arrowsmith et al., 11 Nature Rev. Drug Discov. 384 (2012). Seealso, U.S. Pat. No. 9,255,097.

Histone “demethylases” are enzymes that remove at least one methyl groupfrom a polypeptide, and particular demethylases may demethylate either amono-, di- or a tri-methylated substrate. Histone demethylases can acton a substrate including methylated core histones, mononucleosomes,dinucleosomes, oligonucleosomes, peptides, or chromatin (e.g., in acell-based assay). Example lysine-specific demethylases includelysine-specific demethylase 1 (LSD1 or KDM1), that demethylates bothmono- and di-methylated H3K4 or H3K9 using flavin as a cofactor; and afamily of demethylases characterized by a ˜150 amino acid-long Jumonji C(JmjC) domain (e.g., jumonji domain-containing histone demethylase 1[JHDM1/KDM2A]). See, e.g., U.S. Pat. No. 9,255,097; WO 2015/200709.

More specifically, human LSD1 and its paralog, LSD2, demethylate bothmono- and di-methylated histone H3 lysine 4 (H3K4) and H3K9 via aFAD-dependent amine oxidation reaction. Unlike the FAD-dependentmechanism of LSD1/2, the Jumonji C domain-containing (JMJD) proteins actthrough a dioxygenase reaction mechanism that requires Fe²⁺, O₂, and2-oxoglutarate to demethylate histones. The JMJD catalytic step is thehydroxylation of a lysine methyl group, which converts it to ahydroxymethyl moiety that spontaneously leaves the nitrogen center ofthe lysine and releases formaldehyde. This reaction allows JMJDproteins, in principle, to demethylate tri-, di- and mono-methylatedlysine residues, whereas LSD1/2 cannot attack trimethylated lysineresidues due to the requirement of a free electron pair on themethylated nitrogen. Most of the JMJD proteins demethylate H3K4, H3K9,H3K27, H3K36, or H4K20, but the enzymatic activity of several JMJDproteins remains unknown, some JMJD demethylases may havemethyl-arginine demethylase activity, and some other JMJD proteins mayhave no catalytic activity at all. See Berry & Janknecht, KDM4/JMJD2Histone Demethylases: Epigenetic Regulators in Cancer Cells, 73(10)Cancer Res. 2936 (2013); see also FIG. 9. In addition to JHDM1/KDM2Amentioned above, JMJD proteins include some thirty human membersphylogenetically clustered into seven subfamilies: JMJD2, JHDM1, JHDM2,JHDM3, JARID, PHF2/PHF8, UTX/UTY, and JmjC domain only. See, e.g., U.S.Pat. No. 9,447,046.

One of the largest JMJD subfamilies, the JMJD2A-E protein family, ispreferentially called KDM4 for K (lysine) demethylase 4. The KDM4subfamily includes KDM4A, B, C, and D. KDM4 can recognize di- andtri-methylated H3K9 and H3K36, as well as trimethylated H1.4K26, assubstrates. Berry & Janknecht, 2013. For example, ectopic expression ofKDM4 family members dramatically decreased levels of tri- anddi-methylated H3K9, and increased levels of mono-methylated H3K9, whichthen delocalized heterochromatin protein 1 and reduced overall levels ofheterochromatin in vivo. Importantly, KDM4 demethylases catalyze thedemethylation of both the repressive H3K9me3 mark and the H3K36me3 mark,the latter linked to transcriptional elongation. Frank et al.,Therapeutic promise of cancer stem cell concept, 120 J. Clin. Invest. 41(2010).

The present embodiments provide use of substituted pyridine derivativecompounds for inhibiting the enzymatic activity of KDM4, andadvantageously treating cancer. In particular, an embodiment of KDM4(i)described herein as Compound I exhibited striking selectivity andefficacy in inhibiting breast cancer tumors, importantly, tumors oftriple-negative breast cancer.

Breast cancer is the worldwide leading cause of cancer death amongwomen. Ferlay et al., GLOBOCAN 2012 v1.0, Cancer Incidence & MortalityWorldwide: IARC Cancer Base No. 11 (2013). Cancer progression isassociated with alterations of epigenetic regulators such ashistone-lysine demethylase 4 (KDM4). Dave & Chang, Treatment resistancein stem cells & breast cancer, 14 J. Mamm. Gland Biol. Neoplasia 79(2009); Frank et al., Therapeutic promise of cancer stem cell concept,120 J. Clin. Invest. 41 (2010); Pattabiraman & Weinberg, TargetingEpithelial-to-Mesenchymal Transition: Case for Differentiation-BasedTherapy, Cold Spring Harbor Sympos. Quantitat. Biol. (2017); Sharma etal., Induction of CXCR2 ligands, stem cell-like phenotype, & metastasisin chemotherapy-resistant breast cancer cells, 372 Cancer Lett. 192(2016).

During breast cancer therapy, classical treatments fail to addressresistant cancer stem-cell populations, and targeting KDM protein orfunction is emerging as a possible route for treatment. Zhang et al.,Cellular orig. & evol. breast cancer, Cold Spring Harbor Perspect. Med.(2017); Chu et al., KDM4B as target for prostate cancer. structuralanalysis & selective inhibition by novel inhibitor, 57 J. Med. Chem.5975 (2014); Labbe et al., Histone lysine demethylase (KDM) subfamily 4.structures, functions & therapeutic potential, 6 Am. J. Transl. Res. 1(2013); Qiu et al., KDM4B & KDM4A promote endometrial cancer progressionby regulating androgen receptor, c-myc, & p27kip1, 6 Oncotarget 31702(2015); Soini et al., KDM4A, KDM4B & KDM4C in non-small cell lungcancer, 8 Int'l J. Clin. Exper. Pathol. 12922 (2015).

The present embodiments provide a KDM4 inhibitor with unique preclinicalcharacteristics. This KDM4(i) is a highly potent pan-KDM4 inhibitor thatspecifically blocks the demethylase activity of KDM4A, B, C, and D butnot that of the other members of the KDM family. The KDM4(i) anti-tumorproperties were validated under conditions recapitulating patienttumors.

Another aspect of the present embodiments provides a method to isolateand grow triple-negative breast cancer stem-cells (BCSCs) fromindividual patient tumors after neoadjuvant chemotherapy. Limitingdilution orthotopic xenografts of these BCSCs faithfully regeneratedoriginal patient tumor histology and gene expression. KDM4(i) blocksproliferation, sphere formation and xenograft tumor growth of BCSCs.Importantly, KDM4(i) abrogates expression of EGFR, a driver oftherapy-resistant, triple-negative breast tumor cells via inhibition ofthe KDM4A demethylase activity. Hsu & Hung, Role of HER2, EGFR, & otherreceptor tyrosine kinases in breast cancer, 35 Cancer Metast. Rev. 575(2016). The present embodiments provide a unique BCSC culture system asa basis for therapeutic compound identification and demonstrate thatKDM4 inhibition is a new therapeutic strategy for the treatment oftriple-negative breast cancer.

Dysregulation of the KDM4 demethylases has been documented in a varietyof cancers including breast cancer. Berry & Janknecht, 2013. It has beenshown that KDM4 controls tumor cell proliferation, particularly inaggressive breast cancers. Ye et al., Genetic alterations of KDM4subfamily & therapeutic effect of novel demethylase inhibitor in breastcancer, 5 Am. J. Cancer. Res. 1519 (2015). Therapy resistance andmetastatic dissemination are the main problems faced during breastcancer treatment. Breast cancer stem-cells have been suggested to beresponsible for both therapy resistance and metastatic dissemination.Chu et al., 2014; Ansieau, EMT in breast cancer stem cell generation,338 Cancer Lett. 63 (2013). These resistant cancer stem-cell (CSC)populations have only been poorly characterized, however, and targetedtherapeutics have yet to be identified. Because KDM4 demethylases mayprovide effective therapeutic targets for the treatment of cancer, ascreen was developed to identify novel KDM4 inhibitors. In order tovalidate inhibitors under conditions that mimic cancer stem-cellpopulations, a new 3D cultivation method was developed as describedherein, using defined serum-free conditions and a low oxygen environmentto enrich BCSCs from individual patient tumors after neoadjuvantchemotherapy.

As shown in Table 1, below, cultures of four different BCSC lines(BCSC1, 2, 3, and 4) were established. All lines originated from primarybreast tumors that were Triple-negative: estrogen receptor (ER),progesterone receptor (PR), and human epidermal growth factor receptor 2(HER2) negative; all xenograft tumors established from these lines weretriple-negative.

TABLE 1 BCSC1-4 patient original tumor, BCSC1-4 cell lines, and BCSC1-4xenografts Patient Tumor Neoadjuvantly administered Xenograft TumorPrimary chemotherapeutic Tumor ID Diagnosis drugs Classificationformation BCSC1 IC FEC, FAC, TAC, TC, Triple-negative 36/38 CisplatinBCSC2 IDC Taxol, Myocet Triple-negative 30/43 BCSC3 IDC EC, TaxotereTriple-negative 7/7 BCSC4 MC AC, Taxol, GemCa Triple-negative 2/2BCSC1-4: breast cancer stem-cells 1-4; IC: invasive carcinoma; IDC:invasive ductal carcinoma; MC: metaplastic carcinoma; FEC:5FU/epi-rubicin/cyclophosphamide; FAC: 5FU/doxorubicin/cyclophosphamide;TAC: docetaxel/doxo-rubicin/cyclophosphamide; TC:docetaxel/cyclophosphamide; EC: epirubicin/cyclophosphamide; AC:doxorubicin/cyclophosphamide; GemCa: carboplatin/gemcitabine.

The estimated frequency of cancer stem-cells in the BCSC cultures wasdetermined (by limiting dilution cell number transplantation) to rangefrom 0.26 to 179 in 1×10⁵ cells, as reflected in Table 2:

TABLE 2 Limiting dilution/xenograft tumor formation Number of cells 1 ×10⁵ 1 × 10⁴ 1 × 10³ Stem-cells/1 × 10⁵ BCSC1 36/38 8/8 3/4 5.1 BCSC230/42 3/4 2/2 1.5 BCSC3 6/6 6/6 6/6 179 BCSC4 5/6 3/6 0/6 0.26

Limiting dilution orthotopic xenografts of these triple-negative BCSCsin immunocompromised NOD/SCID mice yielded triple-negative tumors thatclosely match the patient's primary tumor both morphologically andphenotypically. Tables 1 and 2; FIG. 1C; FIG. 5A. Immunohistochemicalanalyses of the mammary epithelial markers cytokeratin 8, E-cadherin,and vimentin, as well as analyses of the proliferation marker Ki67 ofBCSC1 and BCSC2 xenograft tumors, showed that the xenograft tumors sharea similar pattern with parental patient tumors. FIG. 1B; FIG. 5B.Indeed, matching the parental tumor, the BCSC xenografts were negativefor expression of ER, PR, and HER2 proteins expression. Table 1, FIG.1C; FIG. 5C.

Additionally, unsupervised hierarchical clustering analysis of RNAmicroarray data showed that the tumor xenografts share a close profilewith the parental tumors, indicating a preservation of the respectivemolecular tumor subtype. FIG. 1D. The BCSC lines clustered separatelyfrom host tumor and xenograft, specifying the unique properties of thesecells. Nevertheless, they clustered within the host tumor subtypedepicting a close correlation between the three entities. FIG. 1D.

The BCSCs were also cultivated in a 3D and 2D environment, growing asspheroids and mainly epithelial clusters, respectively. FIG. 1E; FIG.5D. When challenged in an anchorage-independent growth assay, BCSC1 andBCSC2 cells demonstrated a sphere-forming capacity of 11% and 17%,respectively. FIG. 1F; FIG. 5E. Unsorted BCSCs, isolated and cultivatedas described above, expressed to varying degrees the well-known CSCmarkers such as CD24/CD44 (FIG. 1G; FIG. 5I) and CD49f/EpCAM (FIG. 1H;FIG. 5J). Taken together, the data show that BCSCs from triple-negativebreast tumors can be isolated and cultivated directly from patienttissue to provide an in vitro cellular platform representing thisdisease. This allows for the identification and validation of novelcancer targeting strategies in vitro and in vivo.

The expression levels in the BCSC cells of the various KDM4 familymembers were evaluated by western blot analysis. Metzger et al., LSD1demethylates repressive histone marks to promoteandrogenreceptor-dependent transcription, 437 Nature 436 (2005). Asshown in FIG. 2A, KDM4A was expressed at high levels in both BCSC1 andBCSC2 cells. These cells also exhibited heterogeneous expression levelsof KDM4B, KDM4C, and KDM4D. These observations indicate that KDM4 aretherapeutic targets for treatment of BCSC populations. Accordingly, ascreen to characterize KDM4(i) was performed to explore compoundssuitable for treating cancers, in particular cancers such as therapyresistant clonal BCSC-originating tumors. This screen confirmed theefficacy of a particular KDM4(i) shown in FIG. 2B (Compound I).Importantly, this KDM4(i) specifically blocked the demethylaseactivities of KDM4A, 4B, 4C, and 4D (IC₅₀<105 nM) but did not affect thedemethylase activity of the other KDMs. Of note, KDM4(i) exhibited aweak effect on the demethylase activity of KDM5B. Table 3 shows thehalf-maximal inhibitory concentration (IC₅₀) of a Compound I against KDMdemethylases:

TABLE 3 KDM4(i) IC₅₀ per KDM4 member KDM4 family member Compound I IC₅₀(μM) KDM4A 0.104 KDM4B 0.056 KDM4C 0.035 KDM4D 0.104 KDM2A >10 KDM2B >10KDM5B 0.750 KDM6A >10 KDM6B >10

Moreover, KDM4(i) strongly inhibited proliferation of several types ofcancer cell lines, including the triple-negative breast cancer cells:MDA-MB-231. Table 4 shows the half-maximal effective concentration(EC₅₀) of Compound I on various cancer cell lines as shown in a 7-daycell MTS assay:

TABLE 4 KDM4(i) EC₅₀ per cancer cell line Cell line Compound I EC₅₀ (nM)Jurkat 1.1 Kyse-150 5.1 MDA-MB-231 5.9 PC-3 8.2 HCT-116 11 Raji 12DU-145 13 HCC-70 27 Kasumi 34 HL-60 43 NCI-H1792 65 NCI-H460 68U-87 >10,000 IR-90 >10,000 ZR-75-1 >10,000

Furthermore, pharmacokinetic studies indicated that KDM4(i) such asCompound I has properties that demonstrate its suitability for use inthe clinic. Example pharmacokinetic data is shown in Table 5:

TABLE 5 KDM4(i) PK parameters Dose iv/po (mg/kg) 5/10 po ½ (hr) 2.57 AUCpo (μg hr/mL) 10.4 Vz (mL/kg) 666 F % 30.4 AUC: Area Under the Curve(plasma concentration-time curve); iv: intravenous; po: by mouth; Vz:apparent volume of distribution during terminal phase; F %:bioavailability (systemic availability of administered dose)

Regarding the efficacy of KDM4(i) on BCSC proliferation, concentrationsas low as 10 nM KDM4(i) (Compound I) inhibited BCSC1 and BCSC2proliferation, and 50 nM KDM4(i) strongly inhibited survival of BCSC1and BCSC2. FIG. 2C, FIG. 2D; FIG. 6A, FIG. 6B. This inhibitory effectwas even more evident in an anchorage-independent sphere formation assayin which KDM4(i) dramatically reduced the sphere-forming capacity ofboth BCSC1 and BCSC2 cells. FIG. 2E; FIG. 6C. Furthermore, when seededunder KDM4(i) treatment in MATRIGEL® matrix, sphere formation of BCSC1cells was diminished significantly in first generation spheres. Whenreseeded after a week under KDM4(i) treatment, secondary sphere-formingcapacity was abolished, even without presence of the inhibitor. FIG. 2F;FIG. 6D. Taken together, the KDM4(i) Compound I exhibited uniquepre-clinical features that support its use for the treatment ofBCSC-driven tumors.

The molecular mechanism of KDM4(i) inhibition was explored further usingtranscriptome analysis that identified the genes differentiallyregulated upon KDM4(i) treatment. More specifically, BCSC1 cellscultivated in the presence and absence of KDM4(i) were analyzed byRNA-seq, which indicated that upon treatment with a KDM4(i), a total of580 genes were differentially regulated. Among them, 254 genes wereupregulated and 326 genes were downregulated. FIG. 3A. Whether thesegenes are direct KDM4A targets was analyzed by ChIP-seq in BCSC1 cellswith an anti-KDM4A antibody. The analyses, shown in FIG. 3B, identified172,692 high-confidence KDM4A peaks. Only 3215 (1.8%) KDM4A locationswere observed in BCSC2 cells treated with siRNA against KDM4A, thusconfirming specificity of the KDM4A antibody. FIG. 7A. This findingprompted the characterization of the KDM4A cistrome intersection withthe KDM4(i) transcriptome. Among the 580 genes differentially regulatedupon treatment with KDM4(i), KDM4A was present at the promoter of 419genes (72%). FIG. 3C. Pathway analysis for these genes revealed an ‘EGFreceptor signaling pathway’ among the top scoring pathways. FIG. 3D.Importantly, the top scoring pathways share a common gene signature of37 direct KDM4A target genes that are differentially regulated upontreatment with KDM4(i). FIG. 3E. Further, qRT-PCR analysis verified thattreatment with KDM4(i) genes reduced the expression levels of genes,such as VCAN, PRR5, ATF4, EGR1, FST, RUNX1, and, importantly, EGFR. FIG.3F.

EGFR is an emerging therapeutic target associated with poor clinicaloutcome of triple-negative breast cancer. Hsu & Hung, 2016. To unravelthe importance of EGFR signaling in growth of BCSC cells, BCSC1 andBCSC2 cells were treated with a specific EGFR inhibitor: erlotinib.Treatment with erlotinib blocked proliferation of both BCSC1 and BCSC2cells. FIG. 7B-FIG. 7E. Furthermore, the 3D-colony forming capacity ofboth BCSC1 and BCSC2 cells was dramatically reduced upon treatment witherlotinib. FIG. 7F, FIG. 7G. Together, these data demonstrated that EGFRcontrols growth of BCSC cells. Importantly, as shown by western blotanalysis, the protein levels of EGFR were dramatically reduced in bothBCSC1 and BCSC2 cells upon treatment with KDM4(i). FIG. 3G; FIG. 7I.Because EGFR is a direct KDM4A target, whether knockdown of KDM4Aaffects EGFR protein levels was determined. As shown,adenoviral-mediated knockdown of KDM4A lead to reduced levels of EGFR inboth BCSC1 and BCSC2 cells. FIG. 3H; FIG. 7I. These data indicate thatin BCSCs, KDM4(i) inhibition of KDM4A blocks EGFR expression.

Because KDM4A is a demethylase of the repressive H3K9me3 mark, uponinactivation of KDM4A by KDM4(i) an increase in H3K9me3 might beobserved. A ChIP-seq assay using an anti-H3K9me3 antibody identified141,722 high-confidence H3K9me3 peaks in untreated cells and 144,266peaks in KDM4(i)-treated cells. FIG. 7J. Overlap of KDM4A locations withH3K9me3 locations in presence and absence of KDM4(i) revealed that81,717 locations were co-occupied. FIG. 7K. A global increase of theH3K9me3 reads over the KDM4A peaks was observed. FIG. 3I. Similarly, onthe EGFR promoter, an increase of the repressive H3K9me3 mark over theKDM4A peak was observed subsequent to inactivation by KDM4(i). FIG. 3J.These data correlate with the transcriptional repression that isobserved upon KDM4(i) treatment. FIG. 3F. In summary, treating BCSCswith KDM4(i) targets EGFR, a main driver of therapy-resistanttriple-negative breast tumor cells, by inhibiting KDM4A demethylaseactivity.

Additionally, the impact of KDM4(i) on the growth of BCSC1 and BCSC2tumor xenografts was studied in immunocompromised NOD/SCID mice carryingbudding xenograft tumors following 21 days of treatment with KDM4(i).Treatment with KDM4(i) strongly affected tumor growth and final tumorweight of both BCSC1 and BCSC2 xenografts. FIG. 4A-FIG. 4E; FIG. 8A-FIG.8E. Importantly, treatment with KDM4(i) never affected the total weightof the mice. FIG. 8F, FIG. 8G. Furthermore, treatment of KDM4(i)affected expression of KDM4A target genes in a similar way as observedin cell culture. FIG. 3F. As shown in FIG. 4F, expression of PRR5, ATF4,EGR1, FST, RUNX1, and EGFR, was affected in BCSC1 xenograft tumors ofmice treated with KDM4(i). Taken together, treatment with KDM4(i)blocked tumor growth in the BCSC xenograft model.

In summary, the present embodiments establish a novel culture methodthat allows for isolation and growth of BCSC lines isolated fromindividual patient tumors after neoadjuvant chemotherapy. Limitingdilution BCSC xenografts faithfully recapitulate parental patient tumorsand BCSCs, BCSC xenografts, and the parental tumors share a highlysimilar transcriptome profile. Therefore, these models are ideal toolsfor identification and validation of novel therapeutics.

At least one embodiment establishes a method of screening KDM4inhibitory activity of a KDM4 inhibitory compound in primary breastcancer stem cells comprising the steps of: (1) obtaining breast tumormaterial; (2) mechanically dissociating the tumor material; (3) treatingthe tumor material with at least one DNAse, dispase, or thermolysin; (4)diluting the tumor material in buffer; (5) straining the dissociatedtreated tumor material to obtain tumor cells; (6) optionally removingred blood cells with lysis buffer; (7) washing the tumor cells in cellculture media; (8) culturing the washed tumor cells in a stem cellenrichment medium comprising a 1:1 ratio of (a) liquid medium and (b)solid matrix, wherein (a) comprises: mammary epithelial basal medium,serum-free supplement, amphotericin, penicillin-streptomycin, epidermalgrowth factor, fibroblast growth factor, heparin, gentamicin, and Rhokinase inhibitor; (9) incubating the stem cell enrichment culture at 37°C. under low oxygen until the enriched cells proliferate as spheres;(10) expanding the population of cells that proliferated as spheres inan expansion medium comprising (a) and (b) at a 98:2 ratio, to obtainexpanded breast cancer stem cells; (11) reculturing expanded breastcancer stem cells in stem cell enrichment medium comprising a KDM4inhibitor, wherein the KDM4 inhibitor inhibits the ability of breastcancer stem cells to proliferate as spheres in comparison with breastcancer stem cells recultured without a KDM inhibitor.

Further, the present embodiments not only support the pursuit of KDM4family members as novel therapeutic targets, but provide at least onenovel KDM4 inhibitor with unique preclinical characteristics that blocksproliferation of BCSCs in vitro and in vivo by targeting the EGFRpathway. Thus, modulation of KDM4 activity is a promising therapeuticstrategy for the treatment of cancers such as, in particular,chemotherapy-resistant breast cancer. Compound I,3-([(1R)-6-[methyl(phenyl)amino]-1,2,3,4-tetrathydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid, is a specific example of a substituted pyridine derivativecompound comprising a disubstituted pyridine ring bearing at the4-position a carboxylic acid, and at the 3-position a substituted aminogroup. This and related substituted pyridine derivatives are provided inWO 2015/200709.

At least one embodiment provides a KDM4(i) compound having the structureof Formula I:

wherein said compound includes stereoisomers and pharmaceuticallyacceptable salts thereof, and wherein

X is O or CH₂, and

R⁶ is N(R¹)(R²) or O(R²), in which

-   -   R¹ is H or C₁-C₆ alkyl, and    -   R² is optionally substituted aryl, heteroaryl, cyclyl, or        heterocyclyl.

In at least one embodiment, the compound is the R stereoisomer.

In at least one embodiment, R¹ is methyl or ethyl.

In at least one embodiment, R² is heteroaryl such as pyridine.

In at least one embodiment, R² is substituted heteroaryl, such aspyridine substituted with alkyl such as methyl, ethyl, or cyclopropyl.

In at least one embodiment, R² is aryl such as phenyl.

In at least one embodiment, R² is substituted aryl, such as phenyl,substituted with halo, such as fluoro or chloro; or substituted withalkyl, such as methyl, propanyl, or cyclopropyl; or substituted withboth halo and alkyl, such as fluoro and methyl; or substituted with anamino or N-containing group such as dimethylamino, azetidinyl; orsubstituted with a alkoxy such as ethoxy, cyclopropylmethoxy,methoxymethyl, difluoromethoxy, or trifluoromethoxy; or substituted withheterocyclyl such as oxanyl.

In at least one embodiment, R² is an indane moiety such as2,3dihydro-1H-indenyl.

In at least one embodiment, X is CH₂ and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is phenyl or pyridinyl. In a specific embodiment, KDM4(i)is 3-([[(1R)-6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid. In a specific embodiment, the KDM4(i) is3-([[(1R)-6-[methyl(pyridin-2-yl)amino]-1,2,3,4-tetrahydro-naphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is CH₂ and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is phenyl substituted with methyl. In a specificembodiment, the KDM4(i) is3-([[(1R)-6-[methyl(4-methylphenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is CH₂ and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is phenyl substituted with dimethylamino. In a specificembodiment, the KDM4(i) is3-([[(1R)-6-[[4-(dimethylamino)phenyl](methyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is CH₂ and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is phenyl substituted with methoxymethyl, ethoxy, ordifluoromethoxy. In a specific embodiment, KDM4(i) is3-([[(1R)-6-[[4-(methoxymethyl)phenyl](methyl)amino]-1,2,3,4-tetrahydro-naphthalen-1-yl]methyl]amino)pyridine-4-carboxylic acid. In a specific embodiment, KDM4(i) is3-([[(1R)-6-[[4-(difluoromethoxy)phenyl](methyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]-methyl]amino)pyridine-4-carboxylic acid. In a specific embodiment, the KDM4(i) is 3([[(1R)-6-[(4-ethoxyphenyl)(methyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylic acid.

In at least one embodiment, X is CH₂ and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is pyridinyl substituted with methyl. In a specificembodiment, KDM4(i) is 3-([[(1R)-6-[methyl-[5-methylpyridin-2-yl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid. In another specific embodiment, KDM4(i) is3-([[(1R)-6-[methyl[6-methylpyridin-2-yl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ isethyl, and R² is phenyl. In a specific embodiment, KDM4(i) is3-([[(4R)-7-[ethyl(phenyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is substituted phenyl. For example, the phenyl issubstituted with chloro or fluoro. In a specific embodiment, KDM4(i) is3-([[(4R)-7-[3-fluorophenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid. In another specific embodiment, KDM4(i) is3-([[(4R)-7-[4-fluorophenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]-methyl]amino)pyridine-4-carboxylicacid. In another specific embodiment, the KDM4(i) is3-([[(4R)-7-[4-chlorophenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylic acid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is substituted phenyl. For example, the phenyl issubstituted with methyl, ethyl, propyl, or cyclopropyl. In a specificembodiment, the KDM4(i) is 3-([[(4R)-7-[methyl(4-methylphenyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid. In a specific embodiment, the KDM4(i) is3-([[(4R)-7-[methyl(4-ethylphenyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid. In a specific embodiment, the KDM4(i) is3-([[(4R)-7-[methyl[4-propan-2-yl)phenyl]amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is pyridinyl substituted with methyl, ethyl, orcyclopropyl. In a specific embodiment, KDM4(i) is3-([[(4R)-7-[methyl(5-methylpyridin-2-yl)amino]-2,3-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid. In a specific embodiment, the KDM4(i) is 3-([[(4R)-7-[methyl(5-methylpyridin-2-yl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid. In a specific embodiment, the KDM4(i) is3-([[(4R)-7-[(5-cyclopropylpyridin-2-yl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylic acid. In another specific embodiment, the KDM4(i)is3-([[(4R)-7-[(4-cyclopropylphenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is substituted phenyl. For example, the phenyl issubstituted with trifluoroethyl. In a specific embodiment, the KDM4(i)is 3-({[(1R)-6-{[4-(1H-imidazol-1-yl)phenyl](methyl)amino}-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is substituted phenyl. For example, phenyl is substitutedwith trifluoromethoxy, difluoroethoxy, or cyclopropylmethoxy. In aspecific embodiment, KDM4(i) is 3-([[(1R)-6-[[4-(tri-fluoromethoxy)phenyl](methyl)amino3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid. In a specific embodiment, KDM4(i) is3-([[(4R)-7-[[4-(difluoromethoxy)phenyl](methyl)-amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid. In a specific embodiment, KDM4(i) is3-({[(1R)-6-{[4-(cyclo-propylmethoxy)phenyl](methyl)amino}-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is substituted phenyl, such as azetidinyl substitutedphenyl. In a specific embodiment, the KDM4(i) is3-([[(4R)-7-[[4-azetidin-1-yl)phenyl](methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is substituted phenyl. For example, the phenyl issubstituted with oxanyl. In a specific embodiment, the KDM4(i) is3-({[(4R)-7-{methyl[4-(oxan-4-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is 2,3-dihydro-1H-indenyl. In a specific embodiment, theKDM4(i) is3-([[(4R)-7-[2,3-dihydro-1H-inden-5-yl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid.

In at least one embodiment, X is CH₂ and R⁶ is O(R²), in which R² isphenyl substituted with fluoro and methyl. In a specific embodiment, theKDM4(i) is3-([[(1R)-6-[2-fluoro-4-methyl-phenoxy)-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid.

The substituted pyridine derivative compounds are prepared by thegeneral synthetic routes described below in Schemes 1-3.

Referring to Scheme 1, above, compound A and an amine compound B aremixed and treated under a variety of conditions to form compound C. Forexample, the mixture of compound A and an amine B can be subjected tomicrowave irradiation in an appropriate solvent, at temperatures rangingfrom 120° C. to 172° C. The ester compound E can be prepared fromcompound C and an alcohol D using a coupling reagent, such as HATU, inthe presence of a base.

Referring to Scheme 2, above, compound F and an aldehyde compound G aremixed and treated under reductive amination conditions to form compoundC. The ester compound E can be prepared from compound C and an alcohol Dusing a coupling reagent, such as HATU, in the presence of a base.

Referring to Scheme 3, above, compound H and an amine compound B aremixed and treated under a variety of conditions to form compound E. Forexample, the mixture of compound H and an amine B can be subjected to aBuchwald reaction under microwave irradiation in an appropriate solvent,at temperatures ranging from 100° C. to 120° C. The ester compound E canbe hydrolyzed to give compound C, using basic conditions such as INaqueous NaOH.

The KGM4(i) compounds described herein may be produced or provided as apharmaceutically acceptable salt. A pharmaceutically acceptable salt ofany one of the substituted pyridine derivative KGM4(i) compounds isintended to encompass any and all pharmaceutically suitable salt forms,including pharmaceutically acceptable salts such as acid and baseaddition salts, as are well-known in the art.

Typically, the substituted pyridine derivative compound exemplified byCompound I is substantially pure, in that it contains less than about5%, or less than about 1%, or less than about 0.1%, of other organicsmall molecules, such as unreacted intermediates or synthesisby-products that are created, for example, in one or more of the stepsof synthesis.

The KGM4(i) compounds described herein typically contain one or moreasymmetric centers and thus give rise to enantiomers, diastereomers, andother stereoisomeric forms that are defined, in terms of absolutestereochemistry, as (R) or (S). Likewise, all possible isomers, as wellas their racemic and optically pure forms, and all tautomeric forms arealso intended to be included. The term “positional isomer” refers tostructural isomers around a central ring, such as ortho-, meta- andpara-isomers around a benzene ring. A “stereoisomer” refers to acompound made up of the same atoms bonded by the same bonds but havingdifferent three-dimensional structures, which are not interchangeable.Stereoisomers can be separated by means and methods known in the art,such as chiral HPLC. Hence, the KGM4(i) compounds provided hereinencompass various stereoisomers and mixtures thereof and includes“enantiomers,” which refers to two stereoisomers whose molecularstructures are non-superimposable mirror images of one another.Additionally, a “tautomer” refers to a molecule wherein a proton shiftfrom one atom of a molecule to another atom of the same molecule ispossible. The KGM4(i) compounds presented herein may, in certainembodiments, exist as tautomers. In circumstances where tautomerizationis possible, a chemical equilibrium of the tautomers may exist, but theexact ratio of the tautomers depends on factors such as physical state,temperature, solvent, and pH.

The KGM4(i) described herein may be produced, obtained, or formulated asa “prodrug.” Prodrugs are compounds that may be inactive whenadministered, but are converted under physiological conditions or byhydrolysis (i.e., in vivo) to a biologically active compound; thusprodrugs are pharmaceutically acceptable precursors of a biologicallyactive compound. Prodrug compounds may offer advantages of solubility,tissue compatibility, or delayed release in a subject. Prodrugs alsorefer to use of covalently bonded carriers that release the activecompound in vivo when such prodrug is administered to the subject.Prodrugs of an active compound may be prepared by modifying functionalgroups present in the active compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent active compound. For example, prodrugs include compounds inwhich a hydroxy, amino, or mercapto group is bonded to any group that,when the prodrug of the active compound is administered to a mammaliansubject, cleaves to form a free hydroxy, free amino or free mercaptogroup, respectively. Examples of prodrugs include acetate, formate, andbenzoate derivatives of alcohol or amine functional groups in the activecompounds. See, e.g., Bundgard, DESIGN OF PRODRUGS, at 7-9, 21-24(Elsevier, Amsterdam, 1985); Higuchi et al., Pro drugs as Novel DeliverySystems, 14 A.C.S. Symposium Series; BIOREVERSIBLE CARRIERS IN DRUGDESIGN (Edward B. Roche (Ed.), Am. Pharm. Assoc. & Pergamon Press,1987).

Accordingly, and as used herein, a reference to KGM4(i), KGM4(i)compound, or Compound I, and the like, includes within that reference apharmaceutically acceptable salt, hydrate, solvate, N-oxide,stereoisomer, tautomer, or prodrug thereof.

In certain embodiments, the substituted pyridine derivative KGM4(i)compound may be administered as a pure compound. In other embodimentsand in general, the KGM4(i) compound is combined with a pharmaceuticallyacceptable carrier (also referred to herein as a pharmaceuticallysuitable (or acceptable) excipient, physiologically suitable (oracceptable) excipient, or physiologically suitable (or acceptable)carrier) selected on the basis of a chosen route of administration andstandard pharmaceutical practice. See, e.g., REMINGTON: SCIENCE &PRACTICE OF PHARMACY 21^(st) Ed. (Gennaro (Ed.) Mack Pub. Co., Easton,Pa., 2005).

Accordingly, provided herein is a pharmaceutical composition comprisingat least one substituted pyridine derivative KGM4(i) compound, or astereoisomer, pharmaceutically acceptable salt, hydrate, solvate,tautomer, or N-oxide thereof, together with one or more pharmaceuticallyacceptable carriers. The carrier(s) (or excipient(s)) is acceptable orsuitable if the carrier is compatible with the other ingredients of thecomposition and not deleterious to the recipient subject. One embodimentprovides a pharmaceutical composition comprising Compound I.

References to “pharmaceutical agent,” “therapeutic agent,”“pharmaceutically active,” “pharmaceutical,” “drug,” “medicament,”“active agent,” “active drug” “active pharmaceutical ingredient,” andthe like, refer in a general sense to substances useful in the medicaland scientific arts, including, for example, drugs, biologics,diagnostic agents (e.g, dyes or contrast agents) or other substancesused for therapeutic, diagnostic, or preventative (e.g., vaccines), orresearch purposes. Example pharmaceutical agents include smallmolecules, chemotherapeutic agents, contrast agents, anesthetics,interfering RNAs, gene vectors, biologics, immunogens, antigens,interferons, polyclonal antibody preparations, monoclonal antibodies,insulins, or combinations of any of these. As noted, a pharmaceuticalcomposition or pharmaceutical formulation may comprise one or moreactive therapeutic agents, or a combination of active and diagnosticagents, etc., typically further comprising a suitable excipient(s).

Further, a pharmaceutical composition as disclosed here may beformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (i.e., topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid (EDTA); bufferssuch as acetates, citrates or phosphates, and agents for the adjustmentof tonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J., US) or phosphate buffered saline (PBS). Inall cases, the composition must be sterile and should be fluid to theextent that easy syringeability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In this regard, the KDM4(i) Compound I was effectively administered viaoral administration in the Examples described herein. Oral compositionsgenerally include an inert diluent or an edible carrier. They can beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches, and the like, can contain any of the followingingredients or compounds of a similar nature: a binder such asmicrocrystalline cellulose, tragacanth gum, or gelatin; an excipientsuch as starch or lactose; a disintegrating agent such as alginic acid,PRIMOJEL® (sodium starch glycolate, DFE pharma), or corn starch; alubricant such as magnesium stearate, calcium stearate, glycerylpalmitostearate, or glyceryl behenate; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavoring agent such as peppermint, methyl salicylate, or orangeflavoring. Therefore, an example pharmaceutical composition can beformulated in suitable oral dosage forms include, for example, tablets,pills, sachets, or capsules of hard or soft gelatin, methylcellulose orof another suitable material easily dissolved in the digestive tract. Insome embodiments, suitable nontoxic solid carriers are used whichinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. See, e.g.,REMINGTON, 2005.

For example, a tablet can be prepared by mixing 48% by weigh of CompoundI, 45% by weight of microcrystalline cellulose, 5% by weight oflow-substituted hydroxypropyl cellulose, and 2% by weight of magnesiumstearate. Tablets can be prepared by direct compression. The totalweight of this example of compressed tablets is maintained at 250-500mg. Oral doses may typically range from about 1.0 mg to about 1000 mg,one to four times, or more, per day.

In one embodiment, the KDM4(i) compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas sustained/controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. For example, the active ingredients can be entrappedin microcapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

Sustained-release preparations can be prepared, and suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT®(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. Such materials can also be obtainedcommercially (e.g., Alza Corp.; Nova Pharm., Inc.). Liposomalsuspensions (including liposomes targeted to infected cells withmonoclonal antibodies) can also be used as pharmaceutically acceptablecarriers. These preparations can be prepared according to methods knownto those skilled in the art.

Oral or parenteral compositions may be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit formsdisclosed here are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

The pharmaceutical compositions or dosage units can be included in acontainer, pack, or dispenser together with instructions foradministration.

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, an immunostimulatoryagent, chemotherapeutic agent, cytokine, antibody, or growth-inhibitoryagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

A “pharmaceutical formulation,” “formulation,” or “pharmaceuticalcomposition” refers to a drug product that includes at least one activeagent and may further include at least one pharmaceutically acceptableexcipient, carrier, buffer, stabilizer, or other material well-known tothose skilled in the art. For example, a typical injectablepharmaceutical formulation includes a parenterally acceptable aqueoussolution which is pyrogen-free and has suitable pH, isotonicity, andstability. Pharmaceutical compositions can have diagnostic, therapeutic,or research utility in various species, such as for example in humanpatients or subjects. In at least one embodiment, a pharmaceuticalcomposition comprises a bromodomain inhibitor and a chemotherapeuticagent such as temozolomide, protein-bound paclitaxel, or romidepsin. Forexample, a bromodomain inhibitor may be4-[2-(cyclopropylmethylamino)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one.The agents and compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described in accepted literature. See, e.g.,REMINGTON: SCIENCE & PRACTICE OF PHARMACY, 22nd Ed. (Lloyd (Ed.),Pharmaceutical Press, London, U K, 2012). Such formulations contain atherapeutically effective amount of an active agent(s) described herein,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the subject.

A pharmaceutical formulation can include a therapeutically effectiveamount of at least one active agent. Such effective amounts can bereadily determined by one of ordinary skill in the art based, in part,on the effect of the administered dosage form, or the combinatorialeffect of an agent and one or more additional active agents, if morethan one agent is used. A therapeutically effective amount of an activeagent can also vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the agent (and oneor more additional active agents) to elicit a desired response in theindividual, e.g., amelioration of at least one condition parameter. Forexample, a therapeutically effective amount of a dosage form can inhibit(lessen the severity of or eliminate the occurrence of), prevent aparticular disorder, or lessen any one of the symptoms of a particulardisorder known in the art or described herein. A therapeuticallyeffective amount may also be one in which any toxic or detrimentaleffects of the active agent or dosage form are outweighed by thetherapeutically beneficial effects.

Pharmaceutical compositions are administered in a manner appropriate tothe condition treated (see below). An appropriate dose and suitableduration and frequency of dose administration can be determined based onthe condition of the subject, the type and severity of the subject'sdisease, the particular form of the active compound, and the method ofadministration. In general, an appropriate dose and treatment regimenprovides active composition(s) in an amount sufficient to providetherapeutic or prophylactic benefit (e.g., an improved clinical outcome,such as more frequent complete or partial remissions, or longerdisease-free or overall survival, or a lessening of symptom severity,see below). Optimal doses are generally determined using experimentalmodels or clinical trials, then adjusted for the body mass, weight, orblood volume of the subject.

Accordingly, the dose of the pharmaceutical composition comprising atleast one substituted pyridine derivative KDM4(i) compound may differdepending upon the condition of the subject (e.g., human patient), suchas stage of the disease, general health status, age, and other factors.

As noted, the KDM4(i) compounds as disclosed here may be administered incombination therapy, i.e., combined with other agents, e.g., therapeuticagents, that are useful for treating pathological conditions ordisorders, such as various forms of cancer, autoimmune disorders andinflammatory diseases. The term “in combination” in this context meansthat the agents are given substantially contemporaneously, eithersimultaneously or sequentially. If given sequentially, at the onset ofadministration of the second compound, the first of the two compounds ispreferably still detectable at effective concentrations at the site oftreatment.

For example, the combination therapy can include one or more antibodiesdisclosed here coformulated with, or coadministered with, one or moreadditional therapeutic agents, e.g., one or more cytokine and growthfactor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, or cytotoxic or cytostaticagents. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.For example, a therapeutic KDM4(i) compound disclosed here may be usedin combination with an antibody and further include those agents thatinterfere at different stages in an inflammatory response. One or moreKDM4(i) compounds described herein may be coformulated with, orcoadministered with, one or more additional agents such as otherchemotherapeutic agents, or biologics such as vaccines, immunotoxins,cytokine or growth factor antagonists (e.g., soluble receptors, peptideinhibitors, small molecules, ligand fusions); antibodies or antigenbinding portions thereof (e.g., antibodies that bind to tumor markers,cytokines or growth factors or their receptors); and anti-inflammatorycytokines or agonists thereof.

In at least one embodiment, a KDM4(i) compound can be co-formulated orcoadministered with at least one additional chemotherapeutic agent. Thechemotherapeutic agent may be a bromodomain inhibitor (see, e.g., WO2015058160; Patent Pub. No. US 20150111885; U.S. Pat. No. 9,034,900), analkylating agent, or a mitotic inhibitor.

Accordingly, an active agent (i.e., KDM4(i) compound) can beadministered to a subject as a monotherapy, or as a combination therapywith another active agent in a combination dosage form, or as anadditional treatment, e.g., another treatment for the same, anassociated, or an additional disorder. For example, a KDM4(i) compoundcan be combined with a chemotherapeutic agent, such as a bromodomaininhibitor, romidepsin, temozolomide, protein-bound paclitaxel, and thelike, in the same formulation, or in a different formulationadministered simultaneously or sequentially. Additionally, combinationtherapy can include administering to the subject (e.g., a human patient)one or more agents (e.g., antibiotics, anti-coagulants,anti-hypertensives, or anti-inflammatory drugs) that provide atherapeutic benefit to subject. In another example, combination therapycan include administering to the subject a KDM4(i) compound and one ormore additional agents that provide therapeutic benefit to a subject whohas cancer, such as triple-negative or refractory breast cancer.Similarly, in another example, combination therapy can includeadministering to the subject a KDM4(i) compound, protein-boundpaclitaxel, or a combination comprising a KDM4(i) compound andpaclitaxel, and one or more additional agents that provide therapeuticbenefit to a subject who has cancer. In other embodiments, an activeagent is administered first in time and an additional active agent(s) isadministered second in time. In some embodiments, one or more additionalactive agents are administered at the same time, but using differentdrug delivery devices or delivery modes, for example, providing forcombination therapy comprising administration of a KDM4(i) compound andtemozolomide, or comprising a KDM4(i) compound and paclitaxel, orcomprising a KDM4(i) compound and romidepsin. In at least oneembodiment, the KDM4(i) compound is Compound I.

Cancers that may be treated with therapy including administration ofKDM4(i) compounds include carcinoma, sarcoma, germ cell tumor, lymphomaor leukemia, blastoma, or other cancers. Carcinomas include epithelialand glandular neoplasms, transitional cell carcinoma, adenoid cysticcarcinoma, insulinoma, hepatocellular carcinoma, cholangiocarcinoma,carcinoid tumor of appendix, linitis plastica, larynx carcinoma,hypopharynx carcinoma, mouth cancer, hypopharyngeal cancer, salivarygland carcinoma, tongue carcinoma, gastric carcinoma, prolactinoma,oncocytoma, hepatocellular carcinoma, kidney parenchyma carcinoma,papillary renal carcinoma, gall bladder carcinoma, bronchial carcinoma,Grawitz tumor, carcinoma of unknown primary site, multiple endocrineadenomas, endometrioid adenoma, adnexal and skin appendage neoplasms,mucoepidermoid neoplasms, cystic, mucinous and serous neoplasms,cystadenoma, pseudomyxoma peritonei, ductal, lobular and medullaryneoplasms, acinar cell neoplasms, complex epithelial neoplasms,Warthin's tumor, thymoma, specialized gonadal neoplasms, sex cordstromal tumor, solid tumor labial carcinoma, granulosa cell tumor,arrhenoblastoma, Sertoli Leydig cell tumor, glomus tumors,paraganglioma, pheochromocytoma, glomus tumor, melanocytic nevus.Sarcomas include Askin's tumor, botryodies, Ewing's sarcoma, Kaposi'ssarcoma, malignant hemangio endothelioma, malignant schwannoma,osteosarcoma, soft tissue sarcomas (including alveolar soft partsarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma,desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma,extraskeletal chondrosarcoma, extraskeletal osteosarcoma,hemangiopericytoma, hemangiosarcoma, lymphangiosarcoma, lympho-sarcoma,malignant fibrous histiocytoma, neurofibrosarcoma, and synovialsarcoma). Lymphoma and leukemia include acute lymphoblastic leukemia,acute myeloid leukemia, hairy cell leukemia, multiple myeloma, chronicmyelogenous leukemia; chronic myeloproliferative disorders; chroniclymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lympho-plasmacytic lymphoma (such as Waldenstrommacroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma,plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chaindiseases, extranodal marginal zone B-cell lymphoma, also called maltlymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma,non-Hodgkin lymphoma (including diffuse large B-cell lymphoma,follicular lymphoma, Mycosis fungoides and the Sezary syndrome, mantlecell lymphoma, diffuse large B-cell lymphoma, primary effusion lymphoma,intravascular large B-cell lymphoma, hepatosplenic T-cell lymphoma,extranodal NK-/T-cell lymphoma), mediastinal (thymic) large B-celllymphoma, T-cell prolymphocytic leukemia, T-cell large granularlymphocytic leukemia, aggressive NK-cell leukemia, adult T-cellleukemia/lymphoma, enteropathy-type T-cell lymphoma, blastic NK-celllymphoma, cutaneous T-cell lymphoma; primary cutaneous CD30-positiveT-cell lymphoproliferative disorders, primary cutaneous anaplastic largecell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-celllymphoma, peripheral T-cell lymphoma, unspecified, anaplastic large celllymphoma, classical Hodgkin lymphomas (nodular sclerosis, mixedcellularity, lymphocyte-rich, lymphocyte depleted or not depleted,nodular lymphocyte-predominant Hodgkin lymphoma), HIV-related lymphoma(e.g., primary effusion lymphoma). Germ cell tumors include withoutlimitation germinoma, dysgerminoma, nongerminomatous germ cell tumor,endodermal sinus turmor, extracranial germ cell tumor; extragonadal germcell tumor, teratoma, polyembryoma, and gonadoblastoma. Blastomasinclude ependymoblastoma, esthesioneuroblastoma, and nephroblastoma.

Other cancers that may be treated with therapies that includeadministration of KDM4(i) compounds include lung cancers such asnon-small cell lung cancer and small cell lung cancer (including smallcell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma,and combined small cell carcinoma), liver cancer, gastric cancer,glioblastoma, head and neck squamous cell carcinoma, myeloma,adrenocortical carcinoma; thyroid cancer (medullary and papillarythyroid carcinoma), renal carcinoma, cervix carcinoma, uterine corpuscarcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma,urinary carcinoma, melanoma, basalioma, teratoma, choroidea melanoma,craniopharyngeoma, osteosarcoma, myosarcoma, and plasmocytoma, analcancer; appendix cancer; atypical teratoid/rhabdoid tumor; bladdercancer; brain tumor (including brain stem glioma, central nervous systematypical teratoid/rhabdoid tumor, central nervous system embryonaltumors, craniopharyngioma, ependymoma, medulloepithelioma, pinealparenchymal tumors of intermediate differentiation, supratentorialprimitive neuroectodermal tumors and pineoblastoma); breast cancer;bronchial tumors; cancer of unknown primary site; carcinoid tumor;central nervous system atypical teratoid/rhabdoid tumor; central nervoussystem embryonal tumors; childhood cancers; endocrine pancreas isletcell tumors; endometrial cancer; extrahepatic bile duct cancer;gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoidtumor; gastrointestinal stromal cell tumor; gastrointestinal stromaltumor (GIST); gestational trophoblastic tumor; head and neck cancer;heart cancer; intraocular melanoma; islet cell tumors; Langerhans cellhistiocytosis; laryngeal cancer; lip cancer; liver cancer; malignantfibrous histiocytoma bone cancer; medulloepithelioma; Merkel cellcarcinoma; metastatic squamous neck cancer with occult primary; multipleendocrine neoplasia syndromes; multiple myeloma; multiple myeloma/plasmacell neoplasm; myelodysplastic syndromes; myeloproliferative neoplasms;nasal cavity cancer; naso-pharyngeal cancer; oral cavity cancer;oropharyngeal cancer; osteosarcoma; other brain and spinal cord tumors;ovarian epithelial cancer; ovarian germ cell tumor; ovarian lowmalignant potential tumor; papillomatosis; paranasal sinus cancer;parathyroid cancer; pelvic cancer; penile cancer; pineal parenchymaltumors of intermediate differentiation; pineoblastoma; pituitary tumor;plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma; primarycentral nervous system (CNS) lymphoma; rectal cancer; renal cancer;respiratory tract cancer; small intestine cancer; squamous neck cancer;supratentorial primitive neuroectodermal tumors; thymic carcinoma;thymoma; thyroid cancer; transitional cell cancer; transitional cellcancer of the renal pelvis and ureter; trophoblastic tumor; uretercancer; urethral cancer; uterine sarcoma; vaginal cancer; vulvar cancer.

Specific examples of cancers associated with hard tumors that may betreated with substituted pyridine derivative KDM4(i) compounds includebreast cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer,colorectal cancer, kidney cancer, pancreatic cancers (such asglucagonoma, gastrinoma, pancreatic neuroendocrine tumor (VIPoma)), bonecancer, ovarian cancer, prostate cancer, esophageal cancer, stomachcancer, oral cancer, nasal cancer, throat cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadeno-carcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, uterine cancer, testicular cancer, bladder carcinoma, epithelialcarcinoma, glioma, brain tumors (such as glioblastoma, glioblastomamultiforme, astrocytoma, meningioma, medulloblastoma and peripheralneuroectodermal tumors), craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma (malignant melanoma, nodular melanoma, dysplasticnevus, lentigo maligna melanoma, superficial spreading melanoma, andmalignant acral lentiginous melanoma), neuroblastoma, andretinoblastoma. In particular, KDM4(i) compounds may be useful intreating EGFR-pathway-associated cancers.

Chemotherapeutic agents are often characterized by functionality,chemical structure, and relationship to another drug. Chemotherapeuticagents include, for example: alkylating agents (e.g., azacitidine,nitrogen mustards: mechlorethamine, chlorambucil, cyclophosphamide(CYTOXAN®), ifosfamide, bendamustine (LEVACT®) and melphalan;nitrosoureas: streptozocin, carmustine (BCNU), lomustine, andbischloroethylnitrosurea; alkyl sulfonates: busulfan, triazines:dacarbazine (DTIC) and temozolomide (TEMODAR®); ethylenimines: thiotepaand altretamine (hexamethylmelamine); platinum drugs (such as cisplatin,carboplatin, satraplatin (JM-216), CI-973, and oxalaplatin);antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP),capecita-bine (XELODA®), cytarabine (ARA-C®), azacitidine, decitabine(DACOGEN®, 5-aza-2′-deoxy-cytidine, a cytidine analog andhypomethylating agent), floxuridine, fludarabine, gemcitabine (GEMZAR®),hydroxyurea, methotrexate, and pemetrexed (ALIMTA®); anthracyclines(e.g., dauno-rubicin (daunomycin, rubidomycin, or cerubidine),doxorubicin (ADRIAMYCIN®), epirubicin, idarubicin, actinomycin-D,bleomycin; mitomycin-C, and mitoxantrone (which also acts as atopoisomerase II inhibitor)); topoisomerase inhibitors (e.g.,topoisomerase I inhibitors: topotecan, irinotecan (CPT-11);topoisomerase II inhibitors: etoposide (VP-16), camptothecin,teniposide, and mitoxantrone); mitotic inhibitors (e.g., taxanes:paclitaxel (TAXOL®) and docetaxel (TAXOTERE®)); epothilones: ixabepilone(IXEMPRA®); vinca alkaloids such as: vinblastine (VELBAN®), vincristine(ONCOVIN®), and vinorelbine (NAVELBINE®); estramustine (EMCYT®); purineor pyrimidine antagonists such as 6-mercaptopurine, 5-fluorouracil,cytarabine, clofarabine, and gemcitabine; cell maturing agents (e.g.,arsenic trioxide and tretinoin); DNA repair enzyme inhibitors (e.g.,podo-phyllotoxines, etoposide, irinotecan, topotecan, and teniposide);enzymes that prevent cell survival (e.g., asparaginase andpegaspargase); corticosteroids (e.g., prednisone, methylprednisolone(SOLUMEDROL®), and dexamethasone (DECADRON®)); HDAC inhibitors (e.g.,romidepsin (ISTODAX®), vorinostat (ZOLINZA®)); other antimetabolitessuch as L-asparaginase (ELSPA®), 2-deoxy-D-glucose, procarbazine(MATULANE®), and bortezomib (VELCADE®); other cytotoxic agents (e.g.,estramustine phosphate, prednimustine, and procarbazine); hormones(e.g., tamoxifen, leuprolide, flutamide, and megestrol; hormone agonistsor antagonists, partial agonists, or partial antagonists); monoclonalantibodies (e.g., gemtuzumab ozogamicin (MYLOTARG®), inotuzumabozogamicin (CMC-544), alemtuzumab, rituximab, and yttrium-90-ibritumomabtiuxetan); immuno-modulators (e.g., thalidomide and lenalidomide);kinase inhibitors such as Bcr-Abl kinase inhibitors (e.g., AP23464,AZD0530, CGP76030, PD180970, SKI-606, imatinib, dasatinib (BMS354825),nilotinib (AMN107), and VX680/MK-0467 (Aurora kinase inhibitor)).

Additional anticancer therapies that may be combined with BET inhibitortherapy include surgery, radiotherapy (e.g., gamma-radiation, neutronbean radiotherapy, electron beam radiotherapy, proton therapy,brachytherapy, and systemic radioactive isotopes), endocrine therapy,biological response modifiers (e.g., interferons, interleukins, andtumor necrosis factor), hyperthermia and cryotherapy, and agents toattenuate any adverse effects (e.g., anti-emetics).

Reference to a chemotherapeutic agent herein applies to thechemotherapeutic agent or its derivatives and accordingly the inventioncontemplates and includes either of these embodiments (agent; agent orderivative(s)). “Derivatives” or “analogs” of a chemotherapeutic agentor other chemical moiety include, but are not limited to, compounds thatare structurally similar to the chemotherapeutic agent or moiety or arein the same general chemical class as the chemotherapeutic agent ormoiety. In some embodiments, the derivative or analog of thechemotherapeutic agent or moiety retains similar chemical or physicalproperty (including, for example, functionality) of the chemotherapeuticagent or moiety.

Administration of a pharmaceutical composition comprising a KDM4(i)compound may replace or augment a previously or currently administeredtherapy. For example, upon treating with one pharmaceutical formulation,administration of an additional active agent(s) can cease or bediminished, e.g., be administered at lower concentrations or with longerintervals between administrations. In some embodiments, administrationof a previous therapy can be maintained. In some embodiments, a previoustherapy is maintained until the level of an active agent reaches a levelsufficient to provide a therapeutic effect. Accordingly, two therapiescan be administered in combination, sequentially, or simultaneously.Moreover, administration of a KDM4(i) in combination with an additionalactive agent may provide a synergistic therapeutic result. Combinedtherapy provided may be administered at once or multiple times atintervals of time. It is understood that the precise dosage and durationof treatment may vary with the age, weight, and condition of the patientbeing treated, and may be determined empirically using known testingprotocols or by extrapolation from in vivo or in vitro test ordiagnostic data. It is further understood that for any particularindividual, specific dosage regimens should be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of theformulations.

The terms “subject” or “patient” as used herein refer to any subject,particularly a mammalian subject, for whom diagnosis, prognosis, ortherapy of a cancer, such as a breast cancer, particularlytriple-negative breast cancer is relevant. The terms “subject” or“patient” may include any human or nonhuman animal as context indicates.

As used herein, “treat,” “treatment,” “treating,” “palliating,”“ameliorating,” or “treatment of” are used interchangeably and refer, ingeneral, therapeutic benefit or prophylactic benefit, e.g., reducing thepotential for disease, reducing the occurrence of disease, or reducingthe severity of disease. For example, treating can refer to the abilityof a therapy when administered to a subject, to prevent further tumorgrowth or malignancy, or to cure or to alleviate at least partially adisease symptom, sign, or cause. These terms refer to an approach forobtaining beneficial or desired results including but not limited totherapeutic benefit or a prophylactic benefit.

“Therapeutic benefit” generally means eradication or amelioration of theunderlying disorder being treated. A therapeutic benefit may also beachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient is still afflicted with the underlying disorder. Accordingly, atherapeutic benefit is not necessarily a cure for a particular cancer,but rather encompasses a result that most typically includesalleviation; increased survival; elimination of a tumor; reduction of asymptom associated with a cancer; prevention or alleviation of asecondary disease, disorder, or condition resulting from the occurrenceof a cancer; or prevention of metastasis. For prophylactic benefit,compositions may be administered to a patient at risk of developing aparticular disease, or to a patient reporting one or more of thephysiological symptoms of a disease, even though a diagnosis of thisdisease has not been made.

Accordingly, “therapeutic agent” as used herein refers to anytherapeutically active substance that is administered to a subject toproduce a desired, usually beneficial, effect. The term therapeuticagent includes, e.g., classical low molecular weight therapeutic agentscommonly referred to as small molecule drugs; and biologics including,but not limited to, antibodies or functionally active portions thereof,peptides, lipids, protein drugs, protein conjugate drugs, fusionproteins, enzymes, nucleic acids, ribozymes, genetic material, viruses,bacteria, eukaryotic cells, and vaccines. A therapeutic agent can alsobe a pro-drug. A therapeutic agent can also be a radioactive isotope. Atherapeutic agent can be an agent activated by a form of energy such aslight or ultrasonic energy, or activated by other circulating moleculesthat can be administered systemically or locally. In addition, thetherapeutic agent can be pharmaceutically formulated.

EXAMPLES Example 1: Breast Cancer Stem-Cell Lines

Patient breast cancer tumor material was obtained from the pathologydepartment of the University Medical Centre Freiburg with patientconsent (Ethics vote 307/13). All primary tumors were from subjects whohad received chemotherapy before tissue collection and were classifiedas triple-negative. All surgeries were conducted by the Department ofObstetrics and Gynecology at the University Medical Centre Freiburg.Tumor tissue specimens for engraftment and paraffin embedding wereobtained simultaneously through pathologists from the tumor bank of theComprehensive Cancer Centre Freiburg. Written informed consent wasobtained from all patients before inclusion in studies described herein.

Primary breast cancer stem cells (BCSC) lines were isolated bymechanical dissociation of the tumor material and enzymatic digestion in5 mL Dulbecco's PBS buffer (DPBS) (GIBCO® media, Thermo FisherScientific Inc., Waltham, Mass., US) supplemented with 6 U DNAse I(MACHERY-NAGEL GmbH & Co. KG, Düren, DE) and 1 mg LIBERASE™ (RocheDiagnostics GmbH, Mannheim, DE) for 1 hr at 37° C. Afterwards, thedigest medium was diluted with 10 ml DPBS and filtered through a cellstrainer (40 μm, Becton Dickenson, Carlsbad, Calif., US), and remainingtissue clumps smashed with a piston from a 2 mL syringe. Followingcentrifugation at 200 g for 5 min, the supernatant was discarded and thecell pellet washed once with MEBM medium (GIBCO). If red blood cellswere visible in the pellet, 1 mL ACK Lysis buffer (GIBCO) was added tothe cell pellet. After 1 min incubation at room temp, up to 6 mL MEBMwas added and the preparation centrifuged at 200 g for 5 min. After thesupernatant was discarded, the pellet was resuspended in 1 mL MEBM andfiltered through a 40 m strainer. Following centrifugation at 200 g for5 min, the supernatant was discarded and remaining cell pellet taken upin mammary stem-cell (MSC, see below) medium. Cells were counted in aNeubauer chamber (hemocytometer), then plated in a 24-well lowattachment plate (CORNING®, Corning, N.Y., US) at 2×10⁴ cells per wellin an ice-cold 1:1 mix of MSC medium and MATRIGEL® matrix (CORNING,#354230). After solidification of MATRIGEL at 37° C. for 30 min, eachwell was topped with 500 μL MSC medium. The cells were cultured at 37°C. under low-oxygen conditions (3% O₂, 5% CO₂, 92% N₂). When cellsproliferated stably in 3D, they were cultured in 2D culture for cellexpansion.

The basis of the MSC (mammary stem-cell) medium described herein is themammary epithelial basal medium (GIBCO, #31331-028), supplemented withB27® serum-free cell culture supplement (GIBCO, #17504-044),amphotericin B (SIGMA-ALDRICH, # A2942), and penicillin-streptomycin(GIBCO, #15140122). This medium was further supplemented with epidermalgrowth factor (f.c. 20 ng/mL, # AF-100-15, PeproTech, Rocky Hill, N.J.,US), heparin (f.c. 4 μg/mL, Sigma-Aldrich # H3149), fibroblast growthfactor (f.c. 20 ng/ml, PeproTech # AF-100-18B), gentamicin (f.c. g/ml,GIBCO #15750-045), and Rho kinase inhibitor (f.c. 500 nM, CALBIOCHEM®#555550, Merck KGaA, Darmstadt, DE) to complete the MSC medium.

To culture the BCSCs as spheres in a 3D environment, 2×10⁴ cells perwell of a 24-well low-attachment plate were seeded in 100 μL of a 1:1mixture of MATRIGEL:MSC medium. After solidification of the MATRIGEL at37° C. for 30 min, the dish was topped up with 500 μL MSC medium. Cellswere grown under low oxygen conditions as described above. 1 mL MSCmedium was added after 2 days. Cells were split weekly using CORNINGDispase for residual MATRIGEL dissolution and ACCUTASE® cell detachmentsolution (Innovative Cell Technologies, Inc., San Diego, Calif., US) forsphere dissociation. Cells were counted via Neubauer chamber.

To expand the BCSCs in a 2D environment, 4×10⁵ cells per 10 cm culturedish were seeded in 2 mL MSC medium containing 2% MATRIGEL (ice-cold).After solidification of the MATRIGEL at 37° C. for 30 min, the dish wastopped up with 8 mL MSC medium. Cells were grown under low oxygenconditions as described above. Medium was changed after 3 days. Cellswere split weekly using ACCUTASE for detachment and counting beforereseeding.

High titer adenoviral preparations were obtained from Vector BioLabs(Malvern, Pa., US). Adenoviral particles were added (in MSC medium) to amultiplicity of infection (MOI) of 300 for BCSC1 cells, and a MOI of 150for BCSC2 cells.

Example 2: In Vitro Assays

For the cancer stem-cell spheroid assay in methylcellulose, cells weredetached by ACCUTASE solution and counted. 3×10³ single BCSC1 and 1×10³single BCSC2 cells were seeded into individual wells of 96-wellultra-low attachment plates (CORNING, #3474) in serum-free MSC mediumcontaining 1% methylcellulose (Sigma-Aldrich, # M0512). After 7 days,all spheres bigger than four cells were counted forsphere-forming-capacity and spheres over 50 m diameter were counted forboth KDM4(i)-treated and control cells.

For the cancer stem-cell spheroid assay in MATRIGEL matrix, cells weredetached by ACCUTASE solution and counted. 1×10³ as triplicates and4×10⁴ single BCSC1 and single BCSC2 cells were seeded in 50% MATRIGELinto individual wells of 96-well ultra-low attachment plates in MSCmedium. Concentrations of KDM4(i) are as indicated in the figures. After7 days, spheres over 50 m diameter were counted for both KDM4(i)-treatedand control cells in the wells with 1×10³ cells. The wells with 4×10⁴cells were split as described and counted; from these 4×10⁴ cells, 1×10³single BCSC1 and single BCSC2 cells were seeded in triplicates asdescribed herein to assess secondary sphere formation without inhibitorpresent.

For dose-response assays, cells were detached by ACCUTASE solutiondissociation and counted. The wells of a black 384-well plate (GreinerBio-One, Monroe, N.C., US) were coated with 10 μL of MSC mediumcontaining 2% MATRIGEL (354230, Corning). After incubation at 37° C. for30 min to solidify the MATRIGEL, 1×10³ single cells were seeded per384-well in 40 μL medium. After 24 hr under normal culture conditions,the KDM4(i) inhibitor (Compound I, Celgene Quanticel Research, Inc.) wasadded in 50 μL to each well to the final indicated concentrations.Following 96 hr of incubation under normal culture conditions, the cellswere washed once with PBS and fixed with ice-cold methanol for at least15 min at −20° C. After another washing step with PBS, cells werestained with DAPI (Sigma-Aldrich) and read-out with the ScanRmicroscope-based imaging platform (Olympus Deutschland GmbH, Hamburg,DE). Total DAPI cell nuclear counts per well were determined.

For the cell proliferation assays, BCSCs tagged with a stableNLS-mCherry fluorescent signal (nuclear localization peptide) were usedfor this assay. Cells were detached by ACCUTASE dissociation andcounted. Each well of a black 384-well plate (Greiner) was coated with10 μL of MSC medium containing 2% MATRIGEL. After incubation at 37° C.for 30 min to solidify the MATRIGEL, 1×10³ single cells/well were seededin 40 μL medium. After 24 hr normal culture, the KDM4(i) was added in 50μL to each well to the final indicated concentrations. Afterwards, thefirst readout with the ScanR microscope-based imaging platform (Olympus)was started, assessing mCherry-fluorescent cell nuclei in nine sectorsof each well under 60% humidity and 5% CO₂. This readout was repeatedevery 24 hr for 7 days. Analysis was done with the ScanR software(Olympus).

For microarray analysis, total RNA was isolated from patient material,xenografts and cells using the GeneMATRIX Universal RNA Purification Kit(Roboklon GmbH, Berlin, DE) according to manufacturer instruction.Isolated RNAs were processed with the AMBION™ WT Expression kit(Thermo-Fisher) as described by the manufacturer and hybridized toILLUMINA® HT-12 v.4 Expression Bead Chips following standard protocol(Illumina, Inc., San Diego, Calif. US). Expression data were processedand quantile normalized using the R/Bioconductor Beadarray package(PMID: 17586828) v2.22. See Dunning et al., Beadarray: R classes &methods for Illumina bead-based data, 23 Bioinformatics 2183 (2007).Only probesets mapping to an EntrezID via the Bioconductor packageilluminaHumanv4.db (v1.26) were considered for further downstreamanalysis. In case of multiple probesets matching the same EntrezID, theprobeset having the respective highest interquartile range across allsamples were selected. The dendrogram (see figures) depicts acomplete-linkage hierarchical clustering based on the Euclidean distancebetween the samples.

Chromatin immunoprecipitation (ChIP) KDM4(i) assays were performedessentially as described. Metzger et al., LSD1 demethylates repressivehistone marks to promote androgen receptor-dependent transcription, 437Nature 436 (2005). BCSC1 cells were cultured for 18 hr in the absence orpresence of 5×10⁻¹⁰ M KDM4(i). Three days before harvesting, cells wereinfected with adenovirus expressing either shRNA against KDM4A orscrambled control shRNA (Ad-GFP-U6-hKDM4AshRNA and Ad-U6-RNAi-GFP,Vector Biolabs) according to manufacturer instructions.Immunoprecipitation was performed with specific antibodies, anti-KDM4A(Schuele Lab. #5766, lot 5766), anti-H3K9me3 (# C15410056, lotA1675-001P, Diagenode), on GammaBind™ G-Sepharose™ (GE-Healthcare).Libraries were prepared from immunoprecipitated DNA according tostandard methods. ChIP-seq libraries were sequenced using a HiSeq 2000(Illumina) and mapped to the hg19 reference genome using Bowtie2software (e.g., Johns Hopkins Univ., Baltimore, Md., US). Langmead etal., Ultrafast & memory-efficient alignment of short DNA sequences tothe human genome, 10 Genome Biol. R25 (2009). Data were further analyzedusing the peak finding algorithm MACS 1.41 (Model-based Analysis ofChIP-Seq) (e.g., Nat'l Center Biotechnol. Info., U.S. Nat'l LibraryMed.) using input as control. Zhang et al., Model-based analysis ofChIP-Seq (MACS), 9 Genome Biol. (2008). All peaks with FDR greater than1% were excluded from further analysis. The uniquely mapped reads wereused to generate the genome-wide intensity profiles, which werevisualized using the Integrative Genomics Viewer (IGV) genome browser(e.g., Broad Inst., Cambridge, Mass. US). Thorvaldsdottir et al.,Integrative Genomics Viewer (IGV): high-performance genomics datavisualization and exploration, 14 Brief Bioinform. 178 (2013). HOMERsoftware (e.g., Univ. Calif. San Diego) was used to annotate peaks, tocalculate overlaps between different peak files, and for motif searches.Heinz et al., Simple combinations of lineage-determining transcriptionfactors prime cis-regulatory elements required for macrophage and B cellidentities, 38 Mol. Cell 576 (2010). The genomic features (promoter,exon, intron, 3′UTR, and intergenic regions) were defined and calculatedusing Reference Sequence (RefSeq) database (e.g., Nat'l Center forBiotechnol. Info., U.S. Nat'l Library Med.) and HOMER software.

To examine the effect of KDM4 inhibition on cell cycle progression andapoptosis or necrosis, BCSC lines 1 and 2 were treated with 50 nMKDM4(i) for different time points (24 hr, 48 hr, 72 hr, and 96 hr).Following drug exposure, 4×10⁵ cells were collected and stained with 50μL propidium iodide (PI) using the PI/RNase solution (Cell SignalingTechnol. #4087), according to manufacturer instruction. Cells wereanalyzed using BD LSR Fortessa and BD FACS Diva Software (BectonDickinson). The percentage of cells in subG1 G0/G1, S and G2/M phaseswere determined from 1×10⁵ ungated cells using FlowJo software v6.

To analyze the expression of established cancer stem-cell markers, cellswere detached and counted as described above. 1×10⁵ cells were washedwith FACS buffer (PBS+1% BSA) and stained for 20 min at room temp in thedark with the following antibodies diluted in FACS buffer: anti-CD24(eBioscience, 46-0247; 1:100), anti-CD44 (eBioscience, 12-0441-81;1:1000), anti-EpCAM (eBioscience, 660 50-9326; 1:100), and anti-CD49f(eBioscience, 46-0495; 1:200). Cells were analyzed using BD LSR Fortessaand BD FACS Diva Software (Becton Dickinson).

Apoptosis was detected using a FITC Annexin V Apoptosis Detection Kit I(BD Bioscience) according to manufacturer instruction. In brief, cellswere collected with 0.05% trypsin-EDTA solution, washed, and diluted to1×10⁶ cells per ml in 1×Binding Buffer. Staining was performed for 15min at room temp in the dark by adding 5 μL FITC-coupled antibodysolution and 5 μL PI to the cells in 100 μL binding buffer. Afterwards,400 μL binding buffer was added, and cells were analyzed using a BD LSRFortessa and BD FACS Diva Software (Becton Dickinson). A total of 1×10⁵cells were counted. Analysis was performed with FlowJo software v6.

Example 3: In Vivo Tumorigenicity Assay in NOD/SCID Mice BearingOrthotopic BC Xenografts

All mouse handling and experiments were performed in accordance ofGerman Animal Welfare regulations and approved by the local authorities(animal protocol G13/114).

NOD/SCID females (4-5 weeks old) were anesthetized using an isofluraneinhalator. A small sagittal incision (no longer than 1.0 cm) on theshaved and sterilized abdomen allowed access to the mammary glands #4 onboth sides. Tumor cells were mixed with 1×10⁶ irradiated fibroblasts(newborn human foreskin fibroblasts (NuFF), p11, Global Stem, GSC-3002)each, and suspended in a 1:1 mixture of Matrigel:MSC medium. The volumeof each transplant was 40 μL per gland, containing defined numbers ofBCSCs and 1×10⁶ fibroblasts. The transplant was injected into themammary fat pad of the #4 gland on both sides of the animal using a 1 mLsyringe with a fine needle. Each transplant was localized distal to thelymph node in the gland. Surgical incisions were sealed by suturing witha 5/0 thread (Ethicon, Z995). Animals were monitored twice weekly foranimal weight and tumor growth, which was determined by caliper. Tumorvolumes were calculated using the formula: 4/3×π×r³.

Tumor size was monitored using ultrasound measurements gathered using asmall animal high resolution ultrasound system (Vevo3100) and transducer(MX550D) with 40 MHz (VisualSonics, Toronto, Canada). For 3D tumormodelling, the transducer was moved along the tumor automatically with astep size of 0.076 mm. Representative pictures showed qualitative tumordifferences as visualized with Vevo LAB v.1.7.1 at beginning and end oftreatment.

For in vivo treatment, KDM4(i) was solved immediately before treatmentin a vehicle consisting of 50% polyethylene glycol (SIGMA) and 50% DPBS(pH=9, Gibco) with sonication (diagenode bioruptor) until a clearsolution was formed. When tumors reached a palpable size of 2 mm indiameter, mice were randomly assigned to different groups (n=8, eachgroup). The inhibitor was administered daily to NOD/SCID mice via oralgavage at 10 mg/kg. Control animals received vehicle only. Animals weremonitored twice weekly for body weight and tumor growth, which wasdetermined by caliper.

Regarding immunohistochemistry, tissue specimens were immediatelyformalin fixed (10%). After formalin fixation and paraffin embedding 2 mthick sections were cut and mounted onto cover slips. All cover slipswere stored for two days at 58° C. in a drying chamber, subsequentlydeparaffinized using xylene and hydrated with ethanol. Human andcorresponding engrafted tumor tissue was stained using ready to useantibodies for the estrogen-receptor protein (monoclonal rabbitanti-human estrogen receptor a, clone EP1, code IR084)progesterone-receptor protein (monoclonal mouse anti-human progesteronereceptor, clone PgR 636, code IR068), HER2 (polyclonal rabbit anti-humanc-erbB-2 oncoprotein, code A0485), Ki-67 (monoclonal mouse anti-humanKi-67 antigen, clone MIB-1, code IR626), Vimentin (monoclonal mouseanti-Vimentin, clone V9, code IR630), E-Cadherin (monoclonal mouseanti-human E-Cadherin, clone NCH-38, code IR059) and for cytokeratin8/18 (monoclonal rabbit anti-human cytokeratin 8/18, clone EP17/EP30,code IR094). For the (host-dependent) horseradish-based peroxidasedetection ENVISION® Flex Peroxidase-Blocking Reagent (DAKO, SM801),ENVISION® Flex+Rabbit (LINKER) (DAKO, K8019) or ENVISION® Flex+Mouse(LINKER) (DAKO, K8021) and ENVISION® Flex/HRP (DAKO, SM802) were used.Counterstaining was performed with hemalum before adding a coverslip. Asinternal positive control, patient-derived physiological mammary glandwas used for ER, PR, Ki-67 (nuclear staining), cytokeratin 8/18, andE-Cadherin (membranous cytoplasmic staining). The mammarygland-surrounding myoepithelial layer was used as internal control forVimentin. For HER2, tissue specimens from HER2 positive breast cancerpatients (Score 3 according to Ref²⁰) were carried for every HER2staining session as external positive control. Triple-negative breastcancer was defined as ER, PR and HER2 negative (score<2) breast cancer.Goldhirsch et al., Personalizing the treatment of women with earlybreast cancer: highlights of the St Gallen Int'l Expert Consensus onPrimary Therapy of Early Breast Cancer 2013, 24 Ann. Oncol. 2206 (2013).

RNA was isolated as described. Metzger et al., Assembly of methylatedKDM1A and CHD1 drives androgen receptor-dependent transcription andtranslocation, 23 Nat. Str'l Mol. Biol. 132 (2016). Quantitative RT-PCRusing the Abgene SYBR Green PCR kit (Invitrogen) was used according tosupplier protocol, using HPRT for normalization and primer sequences forHPRT. Id. Primers for VCAN, PRR5, ATF4, EGR1, FST, EGFR, RUNX1 are shownin the following table:

SEQ ID Primer Primer Sequences NO: VCAN 5′-ACTGTGGATGGGGTTGTGTT-3′ NO: 15′-CTGCGTCACACTGCTCAAAT-3′ NO: 2 PRR5 5′-CGGGACAAGATTCGCTTCTA-3′ NO: 35′-AGCGCATCCTCTAGCTTCAC-3′ NO: 4 ATF4 5′-CCAACAACAGCAAGGAGGAT-3′ NO: 55′-GTGTCATCCAACGTGGTCAG-3′ NO: 6 EGR1 5′-TGACCGCAGAGTCTTTTCCT-3′ NO: 75′-CACAAGGTGTTGCCACTGTT-3′ NO: 8 FST 5′-GGAAAACCTACCGCAATGAA-3′ NO: 95′-GAGCTGCCTGGACAGAAAAC-3′ NO: 10 EGFR 5′-CCAACCAAGCTCTCTTGAGG-3′ NO: 115′-GCTTTCGGAGATGTTGCTTC-3 NO: 12 RUNX1 5′-CACTGCCTTTAACCCTCAGC-3′ NO: 135′-ACAGAAGGAGAGGCAATGGA-3′ NO: 14

Before harvesting for RNA sequencing (RNA-seq), BCSC1 cells werecultured for 18 hr in the absence or presence of 5×10⁻¹⁰ M KDM4(i) asindicated. RNA samples were sequenced by the standard Illumina protocolto create raw sequence files (.fastq files) at the sequencing corefacility of the DKFZ. These reads were aligned to the hg19 build of thehuman genome using TopHat version 2. The aligned reads were counted withthe Homer software (analyze RNA) and DEG's were identified using EdgeRand DESeq version 1.8.3. Data are deposited under GSE.

Example 4: Chemical Synthesis of Substituted Pyrimidine Derivatives

Unless otherwise noted, reagents and solvents were used as received fromcommercial suppliers. Anhydrous solvents and oven-dried glassware wereused for synthetic transformations sensitive to moisture and/or oxygen.Yields were not optimized. Reaction times are approximate and were notoptimized. Column chromatography and thin layer chromatography (TLC)were performed on silica gel unless otherwise noted. Spectra are givenin ppm (δ) and coupling constants, J are reported in Hertz. For protonspectra, the solvent peak was used as the reference peak. Chemicalsynthesis of3-({[6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylic acid (Compound I) can be carried out, for example,based on the method described in U.S. Pat. No. 9,447,046. Briefly,Compound I can be prepared according to the following preparations:

Preparation 1a: 6-bromo-1,2,3,4-tetrahydronaphthalen-1-one

A solution of NaNO₂ (2.35 g, 34 mmol) in water (10 mL) was addeddropwise to the solution of 6-amino-1,2,3,4-tetrahydronaphthalen-1-one(5.0 g, 31 mmol) in 25% HBr (16 mL) at 0° C. The suspension was thentransferred to a stirred mixture of CuBr (8.9 g, 62 mmol) in 48% HBr (30mL) at 0° C. The resulting mixture was allowed to warm to room temp andstirred for 1 hr. The mixture was extracted with EtOAc, dried (Na₂SO₄),and concentrated. The residue was purified by silica gel chromatography(0%-60% EtOAc/Hex) to give 5.6 g (80%) of the title compound as a lightyellow oil. ¹H NMR (400 MHz, CDCl3): δ 2.10-2.16 (2H, m), 2.64 (2H, t,J=6.4 Hz), 2.94 (2H, t, J=6.0 Hz), 7.42 (1H, s), 7.44 (1H, s), 7.87 (1H,d, J=8.9 Hz). [M+H] calculated for C₁₀H₉BrO: 225, 227. found: 225, 227.

Preparation 1b:6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-one

To a solution of 6-bromo-1,2,3,4-tetrahydronaphthalen-1-one (2.0 g, 8.9mmol) in toluene (20 mL) was added N-methylaniline (960 mg, 8.9 mmol),Cs₂CO₃ (4.4 g, 13.4 mmol), BINAP (310 mg, 0.5 mmol) and Pd(OAc)₂ (110mg, 0.5 mmol). The mixture was stirred overnight at 100° C. under N₂.The mixture was filtered and concentrated, and the residue was purifiedby silica gel chromatography (30%-80% EtOAc/Hex) to give 1.52 g (68%) ofthe title compound as a light brown oil. [M+H] calculated for C₁₇H₁₇NO:252. found: 252.

Preparation 1c:5-(aminomethyl)-N-methyl-N-phenyl-7,8-dihydronaphthalen-2-amine,Hydrochloride

To a solution of Preparation b (0.52 g, 6.0 mmol) and ZnI2 (150 mg) intoluene (20 mL) was added TMSCN (1.2 g, 12 mmol) at room temp. Themixture was heated at 60° C. for 2 hr, then cooled to room temp anddiluted with addition of THF (20 mL). A solution of LAH (5 mL, 2.4 M inTHF, 12 mmol) was added slowly at room temp, and the solution stirredfor 0.5 hr. The reaction was quenched with the addition of EtOAc (10mL), and then water (1 mL) and aqueous 1 M NaOH (1 mL). The solution wasdried (Na₂SO₄) and concentrated to give 1.52 g (89%) of the crude1-(aminomethyl)-6-[methyl(phenyl)amino]-1,2,3,4-tetrahydro-naphthalen-1-olas a white solid.

Into a solution of this intermediate (1.52 g, 5.4 mmol) in methanol (20mL) was bubbled dry HCl gas for 2 min while cooled to maintain the r×ntemp at ≤30° C. The mixture was then stirred at room temp for 1 hr. Themethanol was evaporated under reduced pressure to give 1.4 g (98%) ofthe title compound as the HCl salt. [M+H] calculated for C₁₈H₂₀N₂: 265.found 265.

Preparation 1d:5-(aminomethyl)-N-methyl-N-phenyl-5,6,7,8-tetrahydronaphthalen-2-amine

To a solution of Preparation 1c (1.4 g, 5.3 mmol) in MeOH (30 mL) andconc. HCl (three drops) was added 10% Pd/C (200 mg) at room temp underN₂. The suspension was stirred at room temp for 16 hr under hydrogen at50 psi. The reaction mixture was filtered through celite, adjusted to pHabout 8-9 with sat. Na₂CO₃, dried (Na₂SO₄), and concentrated to give 830mg (59%) of the title compound as a yellow oil. [M+H] calculated forC₁₈H₂₂N₂: 267. found: 267.

Preparation 1e: methyl3-[({6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl}methyl)amino]pyridine-4-carboxylate

To a solution of Preparation Id (500 mg, 1.88 mmol) in DMA (12 mL) wasadded methyl 3-fluoroisonicotinate (300 mg, 1.93 mmol). The reactionmixture was stirred at 170° C. for 1 hr in a microwave, then poured intowater and extracted with EtOAc. Organics were washed with brine, dried(Na₂SO₄), and concentrated. The residue was purified by silica gelchromatography (20%-80% EtOAc/Hex) to give 200 mg (26%) of the titlecompound as a yellow oil. [M+H] calculated for C₂₅H₂₇N₃O₂: 402. found:402.

Preparation 1f: methyl 3-({[(1S)-6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylate

and Preparation 2f: methyl3-({[(1R)-6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylate

Preparation 1e (200 mg) was separated by chiral HPLC (Column: ChiralcelIA, 250 mm*4.6 mm 5 m; Mobile phase: Hex:EtOH=85:15; F: 1.0 mL/min; W:230 nm; T=30° C.) to give 95 mg (47%) of Preparation 1f (6.54 min) and92 mg (46%) of Preparation 2f (7.91 min), each as a yellow oil.

Preparation 1g:3-({[(1S)-6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylicacid

To a solution of Preparation 1f (95 mg, 0.24 mmol) in THF (6 mL) and H₂O(2 mL) was added LiOH.H₂O (31 mg, 0.72 mmol) at room temp, and thereaction mixture was stirred overnight. The reaction mixture wasconcentrated to remove THF, and the residue was diluted with water andacidified to pH about 3-4 with 1.0 N aqueous HCl solution. Theprecipitate was collected by filtration and washed with EtOAc/ether. Thesolid was dried under vacuum to give 52 mg (56%) of the title compoundas a yellow solid. ¹H NMR (400 MHz, DMSO-d6): δ 1.64-1.67 (1H, m),1.77-1.84 (3H, m), 2.65-2.68 (2H, d, J=5.6 Hz), 3.04-3.07 (1H, m), 3.21(3H, s), 3.41-3.47 (1H, m), 3.56-3.60 (1H, m), 6.78-6.92 (5H, m),7.21-7.25 (3H, m), 7.55 (1H, d, J=5.2 Hz), 7.82 (1H, d, J=5.2 Hz), 8.36(1H, s). [M+H] Calculated for C₂₄H₂₅N₃O₂: 388. found: 388.

Preparation 2g:3-({[(1R)-6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylicacid (Compound I)

The title compound was prepared in 53% yield from Preparation 2faccording to the procedure for Preparation 1g. ¹H NMR (400 MHz,DMSO-d6): δ 1.64-1.68 (1H, m), 1.77-1.84 (3H, m), 2.65-2.68 (2H, d,J=5.6 Hz), 3.04-3.07 (1H, m), 3.21 (3H, s), 3.41-3.47 (1H, m), 3.56-3.60(1H, m), 6.78-6.92 (5H, m), 7.21-7.25 (3H, m), 7.56 (1H, d, J=4.8 Hz),7.82 (1H, d, J=5.2 Hz), 8.36 (1H, s). [M+H] calculated for C₂₄H₂₅N₃O₂:388. found: 388.

We claim:
 1. A compound having the structure of Formula I:

wherein said compound includes stereoisomers and pharmaceuticallyacceptable salts thereof, and wherein X is O or CH₂, and R⁶ is N(R¹)(R²)or O(R²), in which R¹ is H or C₁-C₆ alkyl, and R² is optionallysubstituted aryl, heteroaryl, cyclyl, or heterocyclyl.
 2. The compoundof claim 1 is R stereoisomer.
 3. The compound of claim 1, wherein R¹ ismethyl or ethyl.
 4. The compound of claim 1, wherein R² is pyridineoptionally substituted with alkyl selected from methyl, ethyl, andcyclopropyl.
 5. The compound of claim 1, wherein R² is phenyl optionallysubstituted with halo selected from fluoro and choloro; phenyloptionally substituted with alkyl selected from methyl, propanyl, andcyclopropyl; phenyl optionally substituted with amino; phenyl optionallysubstituted with a N-containing group selected from dimethylamino andazetidinyl; phenyl optionally substituted with alkoxy selected fromethoxy, cyclopropylmethoxy, methoxymethyl, difluoromethoxy, andtrifluoromethoxy; or phenyl optionally substituted with heterocyclylselected from azetidinyl and oxanyl.
 6. The compound of claim 1, whereinR² is an indane moiety.
 7. The compound of claim 6, wherein R² is2,3-dihydro-1H-indenyl.
 8. The compound of claim 1, wherein X is CH₂ andR⁶ is N(R¹)(R²), in which R¹ is methyl, and R² is phenyl or pyridinyl.9. The compound of claim 1, wherein X is CH₂ and R⁶ is N(R¹)(R²), inwhich R¹ is methyl, and R² is phenyl substituted with methyl.
 10. Thecompound of claim 1, wherein X is CH₂ and R⁶ is N(R¹)(R²), in which R¹is methyl, and R² is phenyl substituted with dimethylamino.
 11. Thecompound of claim 1, wherein X is CH₂ and R⁶ is N(R¹)(R²), in which R¹is methyl, and R² is phenyl substituted with methoxymethyl, ethoxy, ordifluoromethoxy.
 12. The compound of claim 1, wherein X is CH₂ and R⁶ isN(R¹)(R²), in which R¹ is methyl, and R² is pyridinyl substituted withmethyl.
 13. The compound of claim 1, wherein X is O and R⁶ is N(R¹)(R²),in which R¹ is ethyl, and R² is phenyl.
 14. The compound of claim 1,wherein X is O and R⁶ is N(R¹)(R²), in which R¹ is methyl, and R² isphenyl substituted with chloro or fluoro.
 15. The compound of claim 1,wherein X is O and R⁶ is N(R¹)(R²), in which R¹ is methyl, and R² isphenyl substituted with methyl, ethyl, propyl, or cyclopropyl.
 16. Thecompound of claim 1, wherein X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is pyridinyl substituted with methyl, ethyl, orcyclopropyl.
 17. The compound of claim 1, wherein X is O and R⁶ isN(R¹)(R²), in which R¹ is methyl, and R² is phenyl substituted withtrifluoroethyl.
 18. The compound of claim 1, wherein X is O and R⁶ isN(R¹)(R²), in which R¹ is methyl, and R² is phenyl substituted withtrifluoromethoxy, difluoroethoxy, or cyclopropylmethoxy.
 19. Thecompound of claim 1, wherein X is O and R⁶ is N(R¹)(R²), in which R¹ ismethyl, and R² is phenyl substituted with azetidinyl.
 20. The compoundof claim 1, wherein X is O and R⁶ is N(R¹)(R²), in which R¹ is methyl,and R² is phenyl substituted with oxanyl.
 21. The compound of claim 1,wherein X is O and R⁶ is N(R¹)(R²), in which R¹ is methyl, and R² is2,3-dihydro-1H-indenyl.
 22. The compound of claim 1, wherein X is CH₂and R⁶ is O(R²), in which R² is phenyl substituted with fluoro andmethyl.
 23. A compound selected from: 3-([[(1R)-6-[methyl(phenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(1R)-6-[methyl(pyridin-2-yl)amino]-1,2,3,4-tetrahydro-naphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(1R)-6-[methyl(4-methylphenyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(1R)-6-[[4-(dimethylamino)phenyl](methyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(1R)-6-[[4-(methoxymethyl)phenyl](methyl)amino]-1,2,3,4-tetrahydro-naphthalen-1-yl]methyl]amino)pyridine-4-carboxylic acid;3-([[(1R)-6-[[4-(difluoromethoxy)phenyl](methyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]-methyl]amino)pyridine-4-carboxylic acid; 3([[(1R)-6-[(4-ethoxyphenyl)(methyl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylic acid; 3-([[(1R)-6-[methyl-[5-methylpyridin-2-yl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(1R)-6-[methyl[6-methylpyridin-2-yl)amino]-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[ethyl(phenyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[3-fluorophenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[4-fluorophenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]-methyl]amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[4-chlorophenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylic acid; 3-([[(4R)-7-[methyl(4-methylphenyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[methyl(4-ethylphenyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[methyl[4-propan-2-yl)phenyl]amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[methyl(5-methylpyridin-2-yl)amino]-2,3-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid; 3-([[(4R)-7-[methyl(5-methylpyridin-2-yl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid; 3-([[(4R)-7-[(5-cyclopropylpyridin-2-yl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylic acid;3-([[(4R)-7-[(4-cyclopropylphenyl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid;3-({[(1R)-6-{[4-(1H-imidazol-1-yl)phenyl](methyl)amino}-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylicacid; 3-([[(1R)-6-[[4-(tri-fluoromethoxy) phenyl](methyl)amino3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid; 3-([[(4R)-7-[[4-(difluoromethoxy)phenyl](methyl)-amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylic acid;3-({[(1R)-6-{[4-(cyclo-propylmethoxy)phenyl](methyl)amino}-1,2,3,4-tetrahydronaphthalen-1-yl]methyl}amino)pyridine-4-carboxylicacid; 3-([[(4R)-7-[[4-azetidin-1-yl)phenyl](methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylic acid;3-({[(4R)-7-{methyl[4-(oxan-4-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylicacid;3-([[(4R)-7-[2,3-dihydro-1H-inden-5-yl)(methyl)amino]-3,4-dihydro-2H-1-benzopyran-4-yl]methyl]amino)pyridine-4-carboxylicacid; and3-([[(1R)-6-[2-fluoro-4-methyl-phenoxy)-1,2,3,4-tetrahydronaphthalen-1-yl]methyl]amino)pyridine-4-carboxylic acid.
 24. A pharmaceutical compositioncomprising a compound of Formula I as described in claim 1, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 25. A pharmaceutical composition comprising acompound as described in claim 23, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient.
 26. A method ofinhibiting a cancer associated with lysine demethylase 4 (KDM4) activity(a KDM4-associated cancer) comprising administering to a patient in needthereof a pharmaceutical composition as described in claim
 24. 27. Amethod of inhibiting a cancer associated with KDM4 activity (aKDM4-associated cancer) comprising administering to a patient in needthereof a pharmaceutical composition as described in claim
 25. 28. Themethod of claim 26, wherein the KDM4-associated cancer istriple-negative breast cancer.
 29. The method of claim 27, wherein theKDM4-associated cancer is triple-negative breast cancer.
 30. A method ofselectively inhibiting lysine demethylase (KDM) activity in a cellcomprising contacting the cell with a compound of Formula I as describedin claim
 1. 31. A method of selectively inhibiting KDM activity in acell comprising contacting the cell with a compound as described inclaim
 23. 32. A formulation comprising a tablet containing 48% by weighof a compound as described in claim 23, 45% by weight ofmicrocrystalline cellulose, 5% by weight of low-substitutedhydroxypropyl cellulose, and 2% by weight of magnesium stearate.
 33. Amethod of screening KDM4 inhibitory activity of a KDM4 inhibitorycompound, in primary breast cancer stem cells comprising the steps ofobtaining breast tumor material; mechanically dissociating the tumormaterial; treating the tumor material with at least one DNAse, dispase,or thermolysin; diluting the tumor material in buffer; straining thedissociated treated tumor material to obtain tumor cells; optionallyremoving red blood cells with lysis buffer; washing the tumor cells incell culture media; culturing the washed tumor cells in a stem cellenrichment medium comprising a 1:1 ratio of (a) liquid medium and (b)solid matrix, wherein (a) comprises: mammary epithelial basal medium,serum-free supplement, amphotericin, penicillin-streptomycin, epidermalgrowth factor, fibroblast growth factor, heparin, gentamicin, and Rhokinase inhibitor; incubating the stem cell enrichment culture at 37° C.under low oxygen until the enriched cells proliferate as spheres;expanding the population of cells that proliferated as spheres in anexpansion medium comprising (a) and (b) at a 98:2 ratio, to obtainexpanded breast cancer stem cells; reculturing expanded breast cancerstem cells in stem cell enrichment medium comprising the KDM4 inhibitorycompound; wherein the KDM4 inhibitory compound inhibits the ability ofbreast cancer stem cells to proliferate as spheres in comparison withbreast cancer stem cells recultured without a KDM4 inhibitor.
 34. Themethod of claim 33, wherein the KDM4 inhibitory compound having thestructure of Formula I of claim 1.